Bcma targetting antibodies, chimeric antigen receptors, and uses thereof

ABSTRACT

Disclosed herein are anti-BCMA antibodies and antigen-binding fragments, chimeric antigen receptors (“CARs”) having these anti-BCMA antibodies and antigen-binding fragments (“BCMA CARs”) and genetically modified immune effector cells having such BCMA CARs. Polynucleotides encoding the anti-BCMA antibodies and antigen-binding fragments and BCMA CARs are also provided herein. Compositions comprising anti-BCMA antibodies and antigen-binding fragments and BCMA CARs are also provided herein. The present disclosure also relates to use of the anti-BCMA antibodies and antigen-binding fragments and genetically modified immune effector cells having such BCMA CARs in cancer treatment.

This application is a continuation of PCT/CN2022/112728, filed Aug. 16,2022, which claims the benefit of PCT application PCT/CN2021/112858,filed Aug. 16, 2021, the contents of both are incorporated herein byreference.

REFERENCE TO A SEQUENCE LISTING

In accordance with 37 CFR § 1.831, the present specification makesreference to a Sequence Listing submitted electronically as a .xml filenamed “545412US_ST26”. The .xml file was generated on Nov. 25, 2022 andis 39,995 bytes in size. The entire contents of the Sequence Listing arehereby incorporated by reference.

1. FIELD

The present invention relates to molecular biology, cell biology, andimmuno-oncology. In particular, provided herein include anti-BCMAantibodies, chimeric antigen receptors (CARs) comprising such anti-BCMAantibodies (“BCMA CARs”), genetically engineered immune effector cellsexpressing such BCMA CARs, and uses thereof in treating tumors orcancers.

2. BACKGROUND

B-cell maturation antigen (BCMA) is a transmembrane glycoproteinexpressed on mature B lymphocytes. The expression of BCMA has beenlinked to several diseases including cancers and infectious diseases.Current therapies targeting BCMA, including BCMA-binding chimericantigen receptors (CARs), and cells expressing such CARs, however, haveonly had limited success. Thus, additional BCMA targeting therapeuticoptions represent unmet needs. The compositions and methods providedherein meet these needs and provide other relative advantages.

3. SUMMARY

Provided herein are antibodies or antigen-binding fragments thereof thatspecifically binds BCMA (e.g., human BCMA), comprising: (a) a lightchain variable region (VL) comprising a light chain CDR1 (VL CDR1), alight chain CDR2 (VL CDR2) and a light chain CDR3 (VL CDR3) have aminoacid sequences of SEQ ID NOs: 1, 2, and 3, respectively; or a variantthereof having up to about 5 amino acid substitutions, additions, and/ordeletions in the VL CDRs; and/or (b) a heavy chain variable region (VH)comprising a heavy chain CDR1 (VH CDR1), a heavy chain CDR2 (VH CDR2),and a heavy chain CDR3 (VH CDR3) have amino acid sequences of SEQ IDNOs: 4, 5, and 6; or a variant thereof having up to about 5 amino acidsubstitutions, additions, and/or deletions in the VH CDRs.

In some embodiments, the antibodies and antigen-binding fragmentsprovided herein comprise a VL CDR1, a VL CDR2, a VL CDR3, a VH CDR1, aVH CDR2 and a VH CDR3, wherein (a) the VL CDR1, CDR2 and CDR3 have theamino acid sequences of SEQ ID NOs: 1, 2, and 3 respectively; and (b)the VH CDR1, CDR2 and CDR3 have the amino acid sequences of SEQ ID NOs:4, 5, and 6 respectively.

Provided herein are antibodies or antigen-binding fragments thereof thatspecifically binds BCMA (e.g., human BCMA), comprising: (a) a VL havingat least 85%, at least 90%, at least 95%, at least 98%, or 100% sequenceidentity to an amino acid sequence of SEQ ID NO:7; and/or (b) a VHhaving at least 85%, at least 90%, at least 95%, at least 98%, or 100%sequence identity to an amino acid sequence of SEQ ID NO:8.

In some embodiments, the antibodies and antigen-binding fragmentsprovided herein comprise a VL and a VH, wherein the VL and VH have theamino acid sequences of SEQ ID NO:7 and 8, respectively.

Provided herein are antibodies or antigen-binding fragments thereof thatspecifically binds BCMA (e.g., human BCMA), comprising (a) a VLcomprising VL CDR1, CDR2, and CDR3 from a VL having an amino acidsequence of SEQ ID NO:7; and/or (b) a VH comprising VH CDR1, CDR2, andCDR3 from a VH having an amino acid sequence of SEQ ID NO: 8.

In some embodiments, provided herein are antibodies or antigen-bindingfragments that compete with an antibody or antigen-binding fragmentdescribed herein for binding to BCMA.

In some embodiments, the antibody or antigen-binding fragment providedherein is a monoclonal antibody or antigen-binding fragment.

In some embodiments, the antibody or antigen-binding fragment providedherein is a bispecific or multispecific antibody.

In some embodiments, the antibody provided herein is a Bi-specificT-cell engager (BiTE).

In some embodiments, the antibody provided herein is selected from thegroup consisting of an IgG1 antibody, an IgG2 antibody, an IgG3antibody, and an IgG4 antibody.

In some embodiments, the antibody or antigen-binding fragment providedherein is selected from the group consisting of a Fab, a Fab′, aF(ab′)₂, a Fv, a scFv, a (scFv)₂, a single domain antibody (sdAb), and aheavy chain antibody (HCAb).

In some embodiments, the antibody or antigen-binding fragment providedherein is a scFv.

In some embodiments, the antibody or antigen-binding fragment providedherein is a chimeric antibody or antigen-binding fragment, a humanizedantibody or antigen-binding fragment, or a human antibody orantigen-binding fragment.

In some embodiments, the antibody or antigen-binding fragment providedherein is a human antibody or antigen-binding fragment.

Also provided herein are polynucleotides encoding the antibody orantigen-binding fragment described herein.

In some embodiments, the polynucleotide is a messenger RNA (mRNA).

Provided herein are vectors comprising the polynucleotide describedherein.

Provided herein are host cells comprising the polynucleotide describedherein or vectors described herein.

Also provided herein are Chimeric Antigen Receptors (CAR) thatspecifically bind BCMA, comprising, from N-terminus to C-terminus: (a) aBCMA-binding domain that comprises an antibody or antigen-bindingfragment described herein; (b) a transmembrane domain; and (c) acytoplasmic domain.

In some embodiments, the transmembrane domain is derived from CD8, CD28,CD3ζ, CD4, 4-1BB, OX40, ICOS, CTLA-4, PD-1, LAG-3, 2B4, BTLA, TCR αchain, TCR β chain, or TCR ζ chain, CD3ε, CD45, CD5, CD8, CD9, CD16,CD22, CD33, CD37, CD64, CD80, CD86, CD134, or CD154.

In some embodiments of the CARs provided herein, the transmembranedomain comprises CD8 transmembrane region or CD28 transmembrane region.

In some embodiments, the cytoplasmic domain comprises a signaling domainderived from CD3ζ, FcRγ, FcγRIIa, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22,CD79a, CD79b, DAP10, DAP12, or any combination thereof.

In some embodiments, the cytoplasmic domain further comprises aco-stimulatory domain derived from CD28, 4-1BB (CD137), OX40, ICOS,DAP10, 2B4, CD27, CD30, CD40, CD2, CD7, LIGHT, GITR, TLR, DR3, CD43, orany combination thereof.

In some embodiments, the cytoplasmic domain comprises a CD3ζ signalingdomain and a 4-1BB co-stimulatory domain.

In some embodiments, the cytoplasmic domain comprises a CD3ζ signalingdomain and a CD28 co-stimulatory domain.

In some embodiments, the CARs provided herein further comprise a CD8hinge between the antibody or antigen-binding fragment and thetransmembrane domain.

In some embodiments, provided herein are CAR that specifically bindsBCMA comprising an amino acid sequence of SEQ ID NO:15.

Also provided herein are polynucleotides encoding a CAR describedherein. In some embodiments, the polynucleotide is a mRNA.

Also provided herein are vectors comprising a polynucleotide describedherein or a vector described herein.

In some embodiments, the cell provided herein is an immune effectorcell.

In some embodiments, the cell is derived from a cell isolated fromperipheral blood or bone marrow.

In some embodiments, the cell is derived from a cell differentiated invitro from a stem or progenitor cell selected from the group consistingof a T cell progenitor cell, a hematopoietic stem and progenitor cell, ahematopoietic multipotent progenitor cell, an embryonic stem cell, andan induced pluripotent cell.

In some embodiments, the cell is a T cell or a NK cell.

In some embodiments, the cell is a cytotoxic T cell, a helper T cell, agamma delta T, a CD4+/CD8+ double positive T cell, a CD4+ T cell, a CD8+T cell, a CD4/CD8 double negative T cell, a CD3+ T cell, a naive T cell,an effector T cell, a helper T cell, a memory T cell, a regulator Tcell, a Th0 cell, a Th1 cell, a Th2 cell, a Th3 (Treg) cell, a Th9 cell,a Th17 cell, a Than helper cell, a Tfh cell, a stem memory TSCM cell, acentral memory TCM cell, an effector memory TEM cell, or an effectormemory TEMRA cell.

In some embodiments, the cell is a cytotoxic T cell.

In some embodiments, provided herein are a population of the cellsdescribed herein, wherein the population of cells are derived fromperipheral blood mononuclear cells (PBMC), peripheral blood leukocytes(PBL), tumor infiltrating lymphocytes (TIL), cytokine-induced killercells (CIK), lymphokine-activated killer cells (LAK), or marrowinfiltrate lymphocytes (MILs).

In some embodiments, provided herein are pharmaceutical compositionscomprising a therapeutically effective amount of the antibody orantigen-binding fragment described herein, and a pharmaceuticallyacceptable carrier. In some embodiments, provided herein arepharmaceutical compositions comprise a therapeutically effective amountof the cell or cell population described herein, and a pharmaceuticallyacceptable carrier.

In some embodiments, provided herein are uses of the antibody orantigen-binding fragment described herein, the cell or population ofcells described herein, or the pharmaceutical composition describedherein in cancer treatment.

In some embodiments, provided herein are use of the antibody orantigen-binding fragment described herein, the cell or population ofcells described herein, or the pharmaceutical composition describedherein for the preparation of a medicament for the treatment of cancer.

In some embodiments, the cell, population of cells, or pharmaceuticalcomposition is used in combination with an additional therapy.

In some embodiments, provided herein are methods of treating cancer in asubject in need thereof, comprising administering to the subject atherapeutically effective amount of the antibody or antigen-bindingfragment of described herein, or the pharmaceutical compositiondescribed herein.

In some embodiments, provided herein are methods of treating cancer in asubject in need thereof, comprising administering to the subject atherapeutically effective amount of the cell or cell populationdescribed herein.

In some embodiments, the population of cells is autologous to thesubject.

In some embodiments, the methods provided herein further compriseobtaining T cells from the subject.

In some embodiments, the methods provided herein further compriseadministering an additional therapy to the subject.

In some embodiments, the subject is a human.

In some embodiments of the uses or methods provided herein, the canceris a BCMA-expressing cancer.

In some embodiments, the cancer is a solid tumor or a hematologicalcancer.

In some embodiments, the cancer is a B cell malignancy.

In some embodiments, the cancer is a lymphoma, a leukemia, or a plasmacell malignancy.

In some embodiments, the caner is multiple myeloma Waldenstrommacroglobulinemia, Hodgkin's lymphoma or non-Hodgkin's lymphoma.

In some embodiments, the caner is MM.

In some embodiments, the MM is non-secretory multiple myeloma, orsmoldering multiple myeloma.

Provided herein are also methods of preparing a cell capable ofexpressing a CAR that specifically binds BCMA, comprising transferringthe polynucleotide described herein into the cell.

In some embodiments, the cell is selected from the group consisting of aT cell, an NK cell, an NKT cell, a macrophage, a neutrophil, and agranulocyte cell.

In some embodiments, the polynucleotide is transferred viaelectroporation.

In some embodiments, the polynucleotide is transferred via viraltransduction.

In some embodiments, the methods provided herein comprise using alentivirus, a retrovirus, an adenovirus, or an adeno-associated virusfor the viral transduction.

In some embodiments, the polynucleotide is transferred using atransposon system.

In some embodiments, the transposon system is Sleeping Beauty orPiggyBac.

In some embodiments, the polynucleotide is transferred usinggene-editing.

In some embodiments, the polynucleotide is transferred using aCRISPR-Cas system, a ZFN system, or a TALEN system.

4. BRIEF DESCRIPTION OF DRAWINGS

FIGS. 1A-1B provides flow cytometry data of anti-BCMA CAR-T cellsstained with CD19-Fc (FIG. 1A) or BCMA-Fc (FIG. 1B).

FIG. 2A provides the frequencies of CAR+ cells of the T cells transducedwith the designated BCMA CARs.

FIG. 2B provides the Medium Fluorescence Intensity (“MFI”) of CARexpression in T cells transduced with the designated BCMA CARs.

FIG. 3 provides the frequencies of CAR+ CD8 cells of the T cellstransduced with the designated BCMA CARs.

FIG. 4 provides the phenotype of designated CART cells characterized byCCR7 expression and CD45RO expression.

FIGS. 5A-5B provide the expression of BCMA in tumor lines. FIG. 5Aprovides the FACS results. FIG. 5B provides the relative expressionlevels as compared to A549 cells.

FIGS. 6A-6B provide ELISA results showing the production of INF-γ andIL-2 by designated CART cells FIG. 6A shows INF-γ production. FIG. 6Bshows IL-2 production.

FIGS. 7A-7D provide results of the tumor killing assay showing thecytolytic activities of designated CART cells against Jeko-1 cells atdifferent E (T cells):T (tumor cells) ratio. FIG. 7A: E:T=0.1:1; FIG.7B: E:T=0.5:1; FIG. 7C: E:T=2:1; FIG. 7D: E:T=2:1 (enlarged view).

FIGS. 8A-8E provide results of the tumor killing assay showing thecytolytic activities of designated CART cells against RPMI-8226 cells.FIG. 8A: E:T=0.1:1; FIG. 8B: E:T=0.5:1; FIG. 8C: E:T=0.5:1 (enlargedview); FIG. 8D: E:T=2:1; FIG. 8D: E:T=2:1 (enlarged view).

5. DETAILED DESCRIPTION

Before the present disclosure is further described, it is to beunderstood that the disclosure is not limited to the particularembodiments set forth herein, and it is also to be understood that theterminology used herein is for the purpose of describing particularembodiments, and is not intended to be limiting.

B-Cell Maturation Protein (BCMA), also known as Tumor Necrosis FactorReceptor Superfamily Member 17 (TNFRSF17) is a member of theTNF-receptor superfamily. BCMA is preferentially expressed in mature Blymphocytes and plays important roles for B cell development andautoimmune response. BCMA has been shown to specifically bind to thetumor necrosis factor (ligand) superfamily, member 13b(TNFSF13B/TALL-1/BAFF), and to lead to NF-kappaB and MAPK8/JNKactivation. BCMA has also been shown to bind to various TRAF familymembers and transduce signals for cell survival and proliferation.

The overexpression and activation of BCMA is associated with humancancer such as multiple myeloma (MM). Shah et al., Leukemia, 34:985-1005(2020). MM is a hematologic malignancy characterized by the uncontrolledproliferation of plasma cells in the bone marrow. About 160,000 newcases are diagnosed, and 110,000 patients die every year in the world.This disease is incurable, although the survival rates are increasingwith the development of new therapeutic approaches.

The present disclosure provides novel antibodies, includingantigen-binding fragments that specifically bind BCMA (e.g., humanBCMA). Further, the present disclosure also provides chimeric antigenreceptors (CARs) that comprise such antibodies or antigen-bindingfragments that specifically bind BCMA (e.g., human BCMA), as well asengineered immune effector cells (e.g., T cells) and populations ofcells that recombinantly express a CAR (e.g., CARTs) that specificallybinds BCMA (e.g., human BCMA). Pharmaceutical compositions comprising atherapeutically effective amount of such antibodies or antigen-bindingfragments, and pharmaceutical compositions comprising a therapeuticallyeffective amount of cells or population of cells are also disclosedherein. Also disclosed herein are uses of such pharmaceuticalcompositions for treating diseases and disorders relating to BCMAexpression (e.g., BCMA expressing cancer) and related methods oftreatment.

5.1 Definitions

Unless otherwise defined herein, scientific and technical terms used inthe present disclosures shall have the meanings that are commonlyunderstood by those of ordinary skill in the art. Further, unlessotherwise required by context, singular terms shall include pluralitiesand plural terms shall include the singular. Generally, nomenclaturesused in connection with, and techniques of, cell and tissue culture,molecular biology, immunology, microbiology, genetics and protein andnucleic acid chemistry and hybridization described herein are thosewell-known and commonly used in the art.

The present disclosure provides novel antibodies, includingantigen-binding fragments that specifically bind BCMA (e.g., humanBCMA). The term “BCMA” includes any variants or isoforms of BCMA whichare naturally expressed by cells. Accordingly, antibodies describedherein can cross-react with BCMA from species other than human (e.g.,cynomolgus BCMA). Alternatively, the antibodies can be specific forhuman BCMA and do not exhibit any cross-reactivity with other species.BCMA or any variants and isoforms thereof, can either be isolated fromcells or tissues which naturally express them or be recombinantlyproduced using well-known techniques in the art and/or those describedherein.

The term “antibody,” and its grammatical equivalents as used hereinrefer to an immunoglobulin molecule that recognizes and specificallybinds a target, such as a protein, polypeptide, peptide, carbohydrate,polynucleotide, lipid, or a combination of any of the foregoing, throughat least one antigen-binding site wherein the antigen-binding site isusually within the variable region of the immunoglobulin molecule. Asused herein, the term encompasses intact polyclonal antibodies, intactmonoclonal antibodies, single-domain antibodies (sdAbs; e.g., camelidantibodies, alpaca antibodies), single-chain Fv (scFv) antibodies, heavychain antibodies (HCAbs), light chain antibodies (LCAbs), multispecificantibodies, bispecific antibodies, monospecific antibodies, monovalentantibodies, and any other modified immunoglobulin molecule comprising anantigen-binding site (e.g., dual variable domain immunoglobulinmolecules) as long as the antibodies exhibit the desired biologicalactivity. Antibodies also include, but are not limited to, mouseantibodies, camel antibodies, chimeric antibodies, humanized antibodies,and human antibodies. An antibody can be any of the five major classesof immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses(isotypes) thereof (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2), basedon the identity of their heavy-chain constant domains referred to asalpha, delta, epsilon, gamma, and mu, respectively. Unless expresslyindicated otherwise, the term “antibody” as used herein include“antigen-binding fragment” of intact antibodies. The term“antigen-binding fragment” as used herein refers to a portion orfragment of an intact antibody that is the antigenic determiningvariable region of an intact antibody. Examples of antigen-bindingfragments include, but are not limited to, Fab, Fab′, F(ab′)2, Fv,linear antibodies, single chain antibody molecules (e.g., scFv), heavychain antibodies (HCAbs), light chain antibodies (LCAbs),disulfide-linked scFv (dsscFv), diabodies, tribodies, tetrabodies,minibodies, dual variable domain antibodies (DVD), single variabledomain antibodies (sdAbs; e.g., camelid antibodies, alpaca antibodies),and single variable domain of heavy chain antibodies (VHH), andbispecific or multispecific antibodies formed from antibody fragments.

The term “humanized antibody” as used herein refers to forms ofnon-human (e.g., murine) antibodies that are specific immunoglobulinchains, chimeric immunoglobulins, or fragments thereof that containminimal non-human sequences. Typically, humanized antibodies are humanimmunoglobulin. In some instances, the Fv framework region residues of ahuman immunoglobulin are replaced with the corresponding residues in anantibody from a non-human species. In some instances, residues of theCDRs are replaced by residues from the CDRs of a non-human species(e.g., mouse, rat, hamster, camel) that have the desired specificity,affinity, and/or binding capability. The humanized antibody can befurther modified by the substitution of additional residues either inthe Fv framework region and/or within the replaced non-human residues torefine and optimize antibody specificity, affinity, and/or bindingcapability. The term “human antibody” as used herein refers to anantibody produced by a human or an antibody having an amino acidsequence corresponding to an antibody produced by a human made using anyof the techniques known in the art.

The term “heavy chain” when used in reference to an antibody refers to apolypeptide chain of about 50-70 kDa, wherein the amino-terminal portionincludes a variable region of about 120 to 130 or more amino acids and acarboxy-terminal portion that includes a constant region. The constantregion can be one of five distinct types, referred to as alpha (a),delta (δ), epsilon (ε), gamma (γ) and mu (μ), based on the amino acidsequence of the heavy chain constant region. The distinct heavy chainsdiffer in size: α, δ and γ contain approximately 450 amino acids, whileμ and ε contain approximately 550 amino acids. When combined with alight chain, these distinct types of heavy chains give rise to five wellknown classes of antibodies, IgA, IgD, IgE, IgG and IgM, respectively,including four subclasses of IgG, namely IgG1, IgG2, IgG3 and IgG4. Aheavy chain can be a human heavy chain.

The term “light chain” when used in reference to an antibody refers to apolypeptide chain of about 25 kDa, wherein the amino-terminal portionincludes a variable region of about 100 to about 110 or more amino acidsand a carboxy-terminal portion that includes a constant region. Theapproximate length of a light chain is 211 to 217 amino acids. There aretwo distinct types, referred to as kappa (κ) of lambda (λ) based on theamino acid sequence of the constant domains. Light chain amino acidsequences are well known in the art. A light chain can be a human lightchain.

The term “variable domain” or “variable region” refers to a portion ofthe light or heavy chains of an antibody that is generally located atthe amino-terminal of the light or heavy chain and has a length of about120 to 130 amino acids in the heavy chain and about 100 to 110 aminoacids in the light chain, and are used in the binding and specificity ofeach particular antibody for its particular antigen. The variabledomains differ extensively in sequence between different antibodies. Thevariability in sequence is concentrated in the CDRs while the lessvariable portions in the variable domain are referred to as frameworkregions (FR). The CDRs of the light and heavy chains are primarilyresponsible for the interaction of the antibody with antigen. Numberingof amino acid positions used herein is according to the EU Index, as inKabat et al. (1991) Sequences of proteins of immunological interest.(U.S. Department of Health and Human Services, Washington, D.C.) 5thed.A variable region can be a human variable region.

A CDR refers to one of three hypervariable regions (H1, H2 or H3) withinthe non-framework region of the immunoglobulin (Ig or antibody) VHβ-sheet framework, or one of three hypervariable regions (L1, L2 or L3)within the non-framework region of the antibody VL β-sheet framework.Accordingly, CDRs are variable region sequences interspersed within theframework region sequences. CDR regions are well known to those skilledin the art and have been defined by a variety of methods/systems. Thesesystems and/or definitions have been developed and refined over yearsand include Kabat, Chothia, IMGT, AbM, and Contact. For example, Kabatdefines the regions of most hypervariability within the antibodyvariable (V) domains (Kabat et al, J. Biol. Chem. 252:6609-6616 (1977);Kabat, Adv. Prot. Chem. 32: 1-75 (1978)). The Chothia definition isbased on the location of the structural loop regions, which defines CDRregion sequences as those residues that are not part of the conservedβ-sheet framework, and thus are able to adapt different conformations(Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). Both terminologiesare well recognized in the art. Additionally, the IMGT system is basedon sequence variability and location within the structure of thevariable regions. The AbM definition is a compromise between Kabat andChothia. The Contact definition is based on analyses of the availableantibody crystal structures. Software programs (e.g., abYsis) areavailable and known to those of skill in the art for analysis ofantibody sequence and determination of CDRs. The positions of CDRswithin a canonical antibody variable domain have been determined bycomparison of numerous structures (Al-Lazikani et al, J. Mol. Biol.273:927-948 (1997); Morea et al, Methods 20:267-279 (2000)). Because thenumber of residues within a hypervariable region varies in differentantibodies, additional residues relative to the canonical positions areconventionally numbered with a, b, c and so forth next to the residuenumber in the canonical variable domain numbering scheme (Al-Lazikani etal., supra (1997)). Such nomenclature is similarly well known to thoseskilled in the art.

For example, CDRs defined according to either the Kabat (hypervariable)or Chothia (structural) designations, are set forth in the table below.

Kabat¹ Chothia² Loop Location VH CDR1 31-35 26-32 linking B and Cstrands VH CDR2 50-65 53-55 linking C′ and C″ strands VH CDR3  95-102 96-101 linking F and G strands VL CDR1 24-34 26-32 linking B and Cstrands VL CDR2 50-56 50-52 linking C′ and C″ strands VL CDR3 89-9791-96 linking F and G strands ¹Residue numbering follows thenomenclature of Kabat et al., supra ²Residue numbering follows thenomenclature of Chothia et al., supra

One or more CDRs also can be incorporated into a molecule eithercovalently or noncovalently to make it an immunoadhesin. Animmunoadhesin can incorporate the CDR(s) as part of a larger polypeptidechain, can covalently link the CDR(s) to another polypeptide chain, orcan incorporate the CDR(s) noncovalently. The CDRs permit theimmunoadhesin to bind to a particular antigen of interest. The CDRregions can be analyzed by, for example, abysis website(http://abysis.org/).

Thus, unless otherwise specified, a CDR, or individual specified CDRs(e.g., VL CDR1, VL CDR2, VL CDR3), of a given antibody or regionthereof, such as a variable region thereof, should be understood toencompass a (or the specific) complementary determining region asdefined by any of the aforementioned schemes, or other known schemes.For example, where it is stated that a particular CDR (e.g., a VH CDR3)contains the amino acid sequence of a corresponding CDR in a given VH orVL region amino acid sequence, it is understood that such a CDR has asequence of the corresponding CDR (e.g., CDR-H3) within the variableregion, as defined by any of the aforementioned schemes, or other knownschemes. In some embodiments, specific CDR sequences are specified,although it is understood that a provided antibody can include CDRs asdescribed according to any of the other aforementioned numbering schemesor other numbering schemes known to a skilled artisan. Likewise, unlessotherwise specified, a FR or individual specified FR(s) (e.g., VH FR1,VH FR2, VH FR3, VH FR4), of a given antibody or region thereof, such asa variable region thereof, should be understood to encompass a (or thespecific) framework region as defined by any of the known schemes.

The terms “epitope” and “antigenic determinant” are used interchangeablyherein an refer to the site on the surface of a target molecule to whichan antibody or antigen-binding fragment binds, such as a localizedregion on the surface of an antigen. The target molecule can comprise, aprotein, a peptide, a nucleic acid, a carbohydrate, or a lipid. Anepitope having immunogenic activity is a portion of a target moleculethat elicits an immune response in an animal. An epitope of a targetmolecule having antigenic activity is a portion of the target moleculeto which an antibody binds, as determined by any method well known inthe art, including, for example, by an immunoassay. Antigenic epitopesneed not necessarily be immunogenic. Epitopes often consist ofchemically active surface groupings of molecules such as amino acids orsugar side chains and have specific three dimensional structuralcharacteristics as well as specific charge characteristics. The term,“epitope” includes linear epitopes and conformational epitopes. A regionof a target molecule (e.g., a polypeptide) contributing to an epitopecan be contiguous amino acids of the polypeptide or the epitope can cometogether from two or more non-contiguous regions of the target molecule.The epitope may or may not be a three-dimensional surface feature of thetarget molecule. Epitopes formed from contiguous amino acids (alsoreferred to as linear epitopes) are typically retained upon proteindenaturing, whereas epitopes formed by tertiary folding (also referredto as conformational epitopes) are typically lost upon proteindenaturing. An epitope typically includes at least 3, and more usually,at least 5, 6, 7, or 8-10 amino acids in a unique spatial conformation.

The term “specifically binds,” as used herein, means that a polypeptideor molecule interacts more frequently, more rapidly, with greaterduration, with greater affinity, or with some combination of the aboveto the epitope, protein, or target molecule than with alternativesubstances, including related and unrelated proteins. A binding moiety(e.g., antibody) that specifically binds a target molecule (e.g.,antigen) can be identified, for example, by immunoassays, ELISAs, SPR(e.g., Biacore), or other techniques known to those of skill in the art.Typically, a specific reaction will be at least twice background signalor noise and can be more than 10 times background. See, e.g., Paul, ed.,1989, Fundamental Immunology Second Edition, Raven Press, New York atpages 332-336 for a discussion regarding antibody specificity. A bindingmoiety that specifically binds a target molecule can bind the targetmolecule at a higher affinity than its affinity for a differentmolecule. In some embodiments, a binding moiety that specifically bindsa target molecule can bind the target molecule with an affinity that isat least 20 times greater, at least 30 times greater, at least 40 timesgreater, at least 50 times greater, at least 60 times greater, at least70 times greater, at least 80 times greater, at least 90 times greater,or at least 100 times greater, than its affinity for a differentmolecule. In some embodiments, a binding moiety that specifically bindsa particular target molecule binds a different molecule at such a lowaffinity that binding cannot be detected using an assay described hereinor otherwise known in the art. In some embodiments, “specifically binds”means, for instance, that a binding moiety binds a molecule target witha K_(D) of about 0.1 mM or less. In some embodiments, “specificallybinds” means that a polypeptide or molecule binds a target with a K_(D)of at about 10 μM or less or about 1 μM or less. In some embodiments,“specifically binds” means that a polypeptide or molecule binds a targetwith a K_(D) of at about 0.1 μM or less, about 0.01 μM or less, or about1 nM or less. Because of the sequence identity between homologousproteins in different species, specific binding can include apolypeptide or molecule that recognizes a protein or target in more thanone species. Likewise, because of homology within certain regions ofpolypeptide sequences of different proteins, specific binding caninclude a polypeptide or molecule that recognizes more than one proteinor target. It is understood that, in some embodiments, a binding moiety(e.g., antibody) that specifically binds a first target may or may notspecifically bind a second target. As such, “specific binding” does notnecessarily require (although it can include) exclusive binding, i.e.,binding to a single target. Thus, a binding moiety (e.g., antibody) can,in some embodiments, specifically bind more than one target. Forexample, an antibody can, in certain instances, comprise two identicalantigen-binding sites, each of which specifically binds the same epitopeon two or more proteins. In certain alternative embodiments, an antibodycan be bispecific and comprise at least two antigen-binding sites withdiffering specificities.

The term “binding affinity” as used herein generally refers to thestrength of the sum total of noncovalent interactions between a bindingmoiety and a target molecule (e.g., antigen). The binding of a bindingmoiety and a target molecule is a reversible process, and the affinityof the binding is typically reported as an equilibrium dissociationconstant (K_(D)). K_(D) is the ratio of a dissociation rate (k_(off) ork_(d)) to the association rate (k_(on) or k_(a)). The lower the K_(D) ofa binding pair, the higher the affinity. A variety of methods ofmeasuring binding affinity are known in the art, any of which can beused for purposes of the present disclosure. Specific illustrativeembodiments include the following. In some embodiments, the “K_(D)” or“K_(D) value” can be measured by assays known in the art, for example bya binding assay. The K_(D) may be measured in a radiolabeled antigenbinding assay (RIA) (Chen, et al., (1999) J. Mol Biol 293:865-881). TheK_(D) or K_(D) value may also be measured by using surface plasmonresonance assays by Biacore, using, for example, a BIAcore™-2000 or aBIAcore™-3000 BIAcore, Inc., Piscataway, N.J.), or by biolayerinterferometry using, for example, the OctetQK384 system (ForteBio,Menlo Park, Calif.).

The term “variant” as used herein in relation to a protein or apolypeptide with particular sequence features (the “reference protein”or “reference polypeptide”) refers to a different protein or polypeptidehaving one or more (such as, for example, about 1 to about 25, about 1to about 20, about 1 to about 15, about 1 to about 10, or about 1 toabout 5) amino acid substitutions, deletions, and/or additions ascompared to the reference protein or reference polypeptide. The changesto an amino acid sequence can be amino acid substitutions. The changesto an amino acid sequence can be conservative amino acid substitutions.A functional fragment or a functional variant of a protein orpolypeptide maintains the basic structural and functional properties ofthe reference protein or polypeptide.

The terms “polypeptide,” “peptide,” “protein,” and their grammaticalequivalents as used interchangeably herein refer to polymers of aminoacids of any length, which can be linear or branched. It can includeunnatural or modified amino acids or be interrupted by non-amino acids.A polypeptide, peptide, or protein can also be modified with, forexample, disulfide bond formation, glycosylation, lipidation,acetylation, phosphorylation, or any other manipulation or modification.

The terms “polynucleotide,” “nucleic acid,” and their grammaticalequivalents as used interchangeably herein mean polymers of nucleotidesof any length and include DNA and RNA. The nucleotides can bedeoxyribonucleotides, ribonucleotides, modified nucleotides or bases,and/or their analogs, or any substrate that can be incorporated into apolymer by DNA or RNA polymerase.

The terms “identical,” percent “identity,” and their grammaticalequivalents as used herein in the context of two or more polynucleotidesor polypeptides, refer to two or more sequences or subsequences that arethe same or have a specified percentage of nucleotides or amino acidresidues that are the same, when compared and aligned (introducing gaps,if necessary) for maximum correspondence, not considering anyconservative amino acid substitutions as part of the sequence identity.The percent identity can be measured using sequence comparison softwareor algorithms or by visual inspection. Various algorithms and softwarethat can be used to obtain alignments of amino acid or nucleotidesequences are well-known in the art. These include, but are not limitedto, BLAST, ALIGN, Megalign, BestFit, GCG Wisconsin Package, and variantsthereof. In some embodiments, two polynucleotides or polypeptidesprovided herein are substantially identical, meaning they have at least70%, at least 75%, at least 80%, at least 85%, at least 90%, and in someembodiments at least 95%, at least 96%, at least 97%, at least 98%, orat least 99% nucleotide or amino acid residue identity, when comparedand aligned for maximum correspondence, as measured using a sequencecomparison algorithm or by visual inspection. In some embodiments,identity exists over a region of the amino acid sequences that is atleast about 10 residues, at least about 20 residues, at least about40-60 residues, at least about 60-80 residues in length or any integralvalue there between. In some embodiments, identity exists over a longerregion than 60-80 residues, such as at least about 80-100 residues, andin some embodiments the sequences are substantially identical over thefull length of the sequences being compared, such as the coding regionof a target protein or an antibody. In some embodiments, identity existsover a region of the nucleotide sequences that is at least about 10bases, at least about 20 bases, at least about 40-60 bases, at leastabout 60-80 bases in length or any integral value there between. In someembodiments, identity exists over a longer region than 60-80 bases, suchas at least about 80-1000 bases or more, and in some embodiments thesequences are substantially identical over the full length of thesequences being compared, such as a nucleotide sequence encoding aprotein of interest.

The term “vector,” and its grammatical equivalents as used herein referto a vehicle that is used to carry genetic material (e.g., apolynucleotide sequence), which can be introduced into a host cell,where it can be replicated and/or expressed. Vectors applicable for useinclude, for example, expression vectors, plasmids, phage vectors, viralvectors, episomes and artificial chromosomes, which can includeselection sequences or markers operable for stable integration into ahost cell's chromosome. Additionally, the vectors can include one ormore selectable marker genes and appropriate expression controlsequences. Selectable marker genes that can be included, for example,provide resistance to antibiotics or toxins, complement auxotrophicdeficiencies, or supply critical nutrients not in the culture media.Expression control sequences can include constitutive and induciblepromoters, transcription enhancers, transcription terminators, and thelike which are well known in the art. When two or more polynucleotidesare to be co-expressed, both polynucleotides can be inserted, forexample, into a single expression vector or in separate expressionvectors. For single vector expression, the encoding polynucleotides canbe operationally linked to one common expression control sequence orlinked to different expression control sequences, such as one induciblepromoter and one constitutive promoter. The introduction ofpolynucleotides into a host cell can be confirmed using methods wellknown in the art. It is understood by those skilled in the art that thepolynucleotides are expressed in a sufficient amount to produce adesired product (e.g., an anti-BCMA antibody or antigen-binding fragmentas described herein), and it is further understood that expressionlevels can be optimized to obtain sufficient expression using methodswell known in the art.

The term “chimeric antigen receptor” or “CAR” as used herein refers toan artificially constructed hybrid protein or polypeptide containing abinding moiety (e.g., an antibody) linked to immune cell (e.g., T cell)signaling or activation domains. In some embodiments, CARs are syntheticreceptors that retarget T cells to tumor surface antigens (Sadelain etal., Nat. Rev. Cancer 3(1):35-45 (2003); Sadelain et al., CancerDiscovery 3(4):388-398 (2013)). CARs can provide both antigen bindingand immune cell activation functions onto an immune cell such as a Tcell. CARs have the ability to redirect T-cell specificity andreactivity toward a selected target in a non-MHC-restricted manner,exploiting the antigen-binding properties of monoclonal antibodies. Thenon-MHC-restricted antigen recognition can give T-cells expressing CARsthe ability to recognize an antigen independent of antigen processing,thus bypassing a mechanism of tumor escape.

The term “genetic engineering” or its grammatical equivalents when usedin reference to a cell is intended to mean alteration of the geneticmaterials of the cell that is not normally found in a naturallyoccurring cell. Genetic alterations include, for example, modificationsintroducing expressible polynucleotides, other additions,mutations/alterations, deletions and/or other functional disruption ofthe cell's genes. Such modifications can be done in, for example, codingregions and functional fragments thereof of a gene. Additionalmodifications can be done in, for example, non-coding regulatory regionsin which the modifications alter expression of a gene.

The term “transfer,” “transduce,” “transfect,” and their grammaticalequivalents as used herein refer to a process by which an exogenouspolynucleotide is introduced into the host cell. A “transferred,”“transfected,” or “transduced” cell is one which has been transferred,transduced, or transfected with an exogenous polynucleotide. The cellincludes the primary subject cell and its progeny. A polynucleotide canbe “transferred” into a host cell using any type of approaches known inthe art, including, e.g., a chemical method, a physical method, or abiological method. A polynucleotide is commonly “transduced” into a hostcell using a virus. By contrast, a polynucleotide is commonly“transfected” into a host cell using a non-viral approach. These termsare used interchangeable at times, and a person of ordinary skill in theart would readily understand their meanings in different contexts.

As used herein, the term “encode” and its grammatical equivalents referto the inherent property of specific sequences of nucleotides in apolynucleotide or a nucleic acid, such as a gene, a cDNA, or an mRNA, toserve as templates for synthesis of other polymers and macromolecules inbiological processes having either a defined sequence of nucleotides(i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and thebiological properties resulting therefrom. Thus, a gene encodes aprotein if transcription and translation of mRNA corresponding to thatgene produces the protein. Unless otherwise specified, a “nucleotidesequence encoding an amino acid sequence” includes all nucleotidesequences that are degenerate versions of each other and that encode thesame amino acid sequence. Nucleotide sequences that encode proteins andRNA can include introns.

A polypeptide, peptide, protein, antibody, polynucleotide, vector, cell,or composition which is “isolated” is a polypeptide, peptide, protein,antibody, polynucleotide, vector, cell, or composition which is in aform not found in nature. Isolated polypeptides, peptides, proteins,antibodies, polynucleotides, vectors, cells, or compositions includethose which have been purified to a degree that they are no longer in aform in which they are found in nature. In some embodiments, apolypeptide, peptide, protein, antibody, polynucleotide, vector, cell,or composition which is isolated is substantially pure.

The term “immune effector cell” and its grammatical equivalents as usedherein and understood in the art refer to cells that are ofhematopoietic origin and play a direct role in the immune responseagainst a target, such as a pathogen, a cancer cell, or a foreignsubstance. Immune effector cells include T cells, B cell, natural killer(NK) cells, NKT cells, macrophages, granulocytes, neutrophils,eosinophils, mast cells, and basophils.

The term “treat” and its grammatical equivalents as used herein inconnection with a disease or a condition, or a subject having a diseaseor a condition refer to an action that suppresses, eliminates, reduces,and/or ameliorates a symptom, the severity of the symptom, and/or thefrequency of the symptom associated with the disease or disorder beingtreated. For example, when used in reference to a cancer or tumor, theterm “treat” and its grammatical equivalents refer to an action thatreduces the severity of the cancer or tumor, or retards or slows theprogression of the cancer or tumor, including (a) inhibiting the growth,or arresting development of the cancer or tumor, (b) causing regressionof the cancer or tumor, or (c) delaying, ameliorating or minimizing oneor more symptoms associated with the presence of the cancer or tumor.

The term “administer” and its grammatical equivalents as used hereinrefer to the act of delivering, or causing to be delivered, atherapeutic or a pharmaceutical composition to the body of a subject bya method described herein or otherwise known in the art. The therapeuticcan be a compound, a polypeptide, an antibody, a cell, or a populationof cells. Administering a therapeutic or a pharmaceutical compositionincludes prescribing a therapeutic or a pharmaceutical composition to bedelivered into the body of a subject. Exemplary forms of administrationinclude oral dosage forms, such as tablets, capsules, syrups,suspensions; injectable dosage forms, such as intravenous (IV),intramuscular (IM), or intraperitoneal (IP); transdermal dosage forms,including creams, jellies, powders, or patches; buccal dosage forms;inhalation powders, sprays, suspensions, and rectal suppositories.

The terms “effective amount,” “therapeutically effective amount,” andtheir grammatical equivalents as used herein refer to the administrationof an agent to a subject, either alone or as a part of a pharmaceuticalcomposition and either in a single dose or as part of a series of doses,in an amount that is capable of having any detectable, positive effecton any symptom, aspect, or characteristics of a disease, disorder orcondition when administered to the subject. The therapeuticallyeffective amount can be ascertained by measuring relevant physiologicaleffects. The exact amount required vary from subject to subject,depending on the age, weight, and general condition of the subject, theseverity of the condition being treated, the judgment of the clinician,and the like. An appropriate “effective amount” in any individual casecan be determined by one of ordinary skill in the art using routineexperimentation.

The term “pharmaceutically acceptable carrier” or “pharmaceuticallyacceptable excipient” refers to a material that is suitable for drugadministration to an individual along with an active agent withoutcausing undesirable biological effects or interacting in a deleteriousmanner with any of the other components of the pharmaceuticalcomposition.

The term “subject” as used herein refers to any animal (e.g., a mammal),including, but not limited to, humans, non-human primates, canines,felines, rodents, and the like, which is to be the recipient of aparticular treatment. A subject can be a human. A subject can have aparticular disease or condition.

The term “autologous” as used herein refers to any material derived fromthe same individual to which it is later to be re-introduced into theindividual.

The term “allogeneic” as used herein refers to a graft derived from adifferent animal of the same species.

Ranges: throughout this disclosure, various aspects of the invention canbe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. Thisapplies regardless of the breadth of the range.

Exemplary genes and polypeptides are described herein with reference toGenBank numbers, GI numbers and/or SEQ ID NOs. It is understood that oneskilled in the art can readily identify homologous sequences byreference to sequence sources, including but not limited to GenBank(ncbi.nlm.nih.gov/genbank/) and EMBL (embl.org/).

5.2 Anti-BCMA Antibodies and Antigen-Binding Fragments

Provided herein are antibodies or antigen-binding fragments thereof thatspecifically bind BCMA (e.g., human BCMA). In some embodiments, providedherein are anti-BCMA antibodies. In some embodiments, the antibody is anIgA, IgD, IgE, IgG, or IgM antibody. In some embodiments, the antibodyis an IgA antibody. In some embodiments, the antibody is an IgDantibody. In some embodiments, the antibody is an IgE antibody. In someembodiments, the antibody is an IgG antibody. In some embodiments, theantibody is an IgM antibody. In some embodiments, the antibodiesprovided herein can be an IgG1 antibody, an IgG2 antibody, an IgG3antibody, or an IgG4 antibody. In some embodiments, the antibody is anIgG1 antibody. In some embodiments, the antibody is an IgG2 antibody. Insome embodiments, the antibody is an IgG3 antibody. In some embodiments,the antibody is an IgG4 antibody.

In some embodiments, provided herein are antigen-binding fragments of ananti-BCMA antibody. In some embodiments, antigen-binding fragmentsprovided herein can be a single domain antibody (sdAb), a heavy chainantibody (HCAb), a Fab, a Fab′, a F(ab′)₂, a Fv, a single-chain variablefragment (scFv), or a (scFv)₂. In some embodiments, the antigen-bindingfragment of an anti-BCMA antibody is a single domain antibody (sdAb). Insome embodiments, the antigen-binding fragment of an anti-BCMA antibodyis a heavy chain antibody (HCAb). In some embodiments, theantigen-binding fragment of an anti-BCMA antibody is a Fab. In someembodiments, the antigen-binding fragment of an anti-BCMA antibody is aFab′. In some embodiments, the antigen-binding fragment of an anti-BCMAantibody is a F(ab′)₂. In some embodiments, the antigen-binding fragmentof an anti-BCMA antibody is a Fv. In some embodiments, theantigen-binding fragment of an anti-BCMA antibody is a scFv. In someembodiments, the antigen-binding fragment of an anti-BCMA antibody is adisulfide-linked scFv [(scFv)₂]. In some embodiments, theantigen-binding fragment of an anti-BCMA antibody is a diabody (dAb).

In some embodiments, the anti-BCMA antibodies or antigen-bindingfragments provided herein comprise recombinant antibodies orantigen-binding fragments. In some embodiments, the anti-BCMA antibodiesor antigen-binding fragments provided herein comprise monoclonalantibodies or antigen-binding fragments. In some embodiments, theanti-BCMA antibodies or antigen-binding fragments provided hereincomprise polyclonal antibodies or antigen-binding fragments. In someembodiments, the anti-BCMA antibodies or antigen-binding fragmentsprovided herein comprise camelid (e.g., camels, dromedary and llamas)antibodies or antigen-binding fragments. In some embodiments, theanti-BCMA antibodies or antigen-binding fragments provided hereincomprise chimeric antibodies or antigen-binding fragments. In someembodiments, the anti-BCMA antibodies or antigen-binding fragmentsprovided herein comprise humanized antibodies or antigen-bindingfragments. In some embodiments, the anti-BCMA antibodies orantigen-binding fragments provided herein comprise human antibodies orantigen-binding fragments. In some embodiments, provided herein areanti-BCMA human scFvs.

In some embodiments, the anti-BCMA antibodies or antigen-bindingfragments provided herein are isolated. In some embodiments, theanti-BCMA antibodies or antigen-binding fragments provided herein aresubstantially pure.

In some embodiments, the anti-BCMA antibody or antigen-binding fragmentprovided herein comprises a multispecific antibody or antigen-bindingfragment. In some embodiments, the anti-BCMA antibody or antigen-bindingfragment provided herein comprises a bispecific antibody orantigen-binding fragment. In some embodiments, provided herein is aBi-specific T-cell engager (BiTE). BiTEs are bispecific antibodies thatbind to a T cell antigen (e.g., CD3) and a tumor antigen. BiTEs havebeen shown to induce directed lysis of target tumor cells and thusprovide great potential therapies for cancers and other disorders. Insome embodiments, provided herein are BiTEs that specifically bind CD3and BCMA. In some embodiments, the BiTEs comprises an anti-BCMA antibodyor antigen-binding fragment provided herein. In some embodiments, theBiTEs comprises an anti-BCMA scFv provided herein.

In some embodiments, the anti-BCMA antibody or antigen-binding fragmentprovided herein comprises a monovalent antigen-binding site. In someembodiments, an anti-BCMA antibody or antigen-binding fragment comprisesa monospecific binding site. In some embodiments, an anti-BCMA antibodyor antigen-binding fragment comprises a bivalent binding site.

In some embodiments, an anti-BCMA antibody or antigen-binding fragmentis a monoclonal antibody or antigen-binding fragment. Monoclonalantibodies can be prepared by any method known to those of skill in theart. One exemplary approach is screening protein expression libraries,e.g., phage or ribosome display libraries. Phage display is described,for example, in Ladner et al., U.S. Pat. No. 5,223,409; Smith (1985)Science 228:1315-1317; and WO 92/18619. In some embodiments, recombinantmonoclonal antibodies are isolated from phage display librariesexpressing variable regions or CDRs of a desired species. Screening ofphage libraries can be accomplished by various techniques known in theart.

In some embodiments, monoclonal antibodies are prepared using hybridomamethods known to one of skill in the art. For example, using a hybridomamethod, a mouse, rat, rabbit, hamster, or other appropriate host animal,is immunized as described above. In some embodiments, lymphocytes areimmunized in vitro. In some embodiments, the immunizing antigen is ahuman protein or a fragment thereof. In some embodiments, the immunizingantigen is a human protein or a fragment thereof.

Following immunization, lymphocytes are isolated and fused with asuitable myeloma cell line using, for example, polyethylene glycol. Thehybridoma cells are selected using specialized media as known in the artand unfused lymphocytes and myeloma cells do not survive the selectionprocess. Hybridomas that produce monoclonal antibodies directed to achosen antigen can be identified by a variety of methods including, butnot limited to, immunoprecipitation, immunoblotting, and in vitrobinding assays (e.g., flow cytometry, FACS, ELISA, SPR (e.g., Biacore),and radioimmunoassay). Once hybridoma cells that produce antibodies ofthe desired specificity, affinity, and/or activity are identified, theclones may be subcloned by limiting dilution or other techniques. Thehybridomas can be propagated either in in vitro culture using standardmethods or in vivo as ascites tumors in an animal. The monoclonalantibodies can be purified from the culture medium or ascites fluidaccording to standard methods in the art including, but not limited to,affinity chromatography, ion-exchange chromatography, gelelectrophoresis, and dialysis.

In some embodiments, monoclonal antibodies are made using recombinantDNA techniques as known to one skilled in the art. For example, thepolynucleotides encoding an antibody are isolated from mature B-cells orhybridoma cells, such as by RT-PCR using oligonucleotide primers thatspecifically amplify the genes encoding the heavy and light chains ofthe antibody, and their sequence is determined using standardtechniques. The isolated polynucleotides encoding the heavy and lightchains are then cloned into suitable expression vectors which producethe monoclonal antibodies when transfected into host cells such as E.coli, simian COS cells, Chinese hamster ovary (CHO) cells, or myelomacells that do not otherwise produce immunoglobulin proteins.

In some embodiments, a monoclonal antibody is modified by usingrecombinant DNA technology to generate alternative antibodies. In someembodiments, the constant domains of the light chain and heavy chain ofa mouse monoclonal antibody are replaced with the constant regions of ahuman antibody to generate a chimeric antibody. In some embodiments, theconstant regions are truncated or removed to generate a desired antibodyfragment of a monoclonal antibody. In some embodiments, site-directed orhigh-density mutagenesis of the variable region(s) is used to optimizespecificity and/or affinity of a monoclonal antibody.

In some embodiments, an anti-BCMA antibody or antigen-binding fragmentis a humanized antibody or antigen-binding fragment. Various methods forgenerating humanized antibodies are known in the art. Methods are knownin the art for achieving high affinity binding with humanizedantibodies. A non-limiting example of such a method is hypermutation ofthe variable region and selection of the cells expressing such highaffinity antibodies (affinity maturation). In addition to the use ofdisplay libraries, the specified antigen (e.g., recombinant BCMA or anepitope thereof) can be used to immunize a non-human animal, e.g., arodent. In certain embodiments, rodent antigen-binding fragments (e.g.,mouse antigen-binding fragments) can be generated and isolated usingmethods known in the art and/or disclosed herein. In some embodiments, amouse can be immunized with an antigen (e.g., recombinant BCMA or anepitope thereof).

In some embodiments, an anti-BCMA antibody or antigen-binding fragmentis a human antibody or antigen-binding fragment. Human antibodies can beprepared using various techniques known in the art. In some embodiments,human antibodies are generated from immortalized human B lymphocytesimmunized in vitro. In some embodiments, human antibodies are generatedfrom lymphocytes isolated from an immunized individual. In any case,cells that produce an antibody directed against a target antigen can begenerated and isolated. In some embodiments, a human antibody isselected from a phage library, where that phage library expresses humanantibodies. Alternatively, phage display technology can be used toproduce human antibodies and antibody fragments in vitro, fromimmunoglobulin variable region gene repertoires from unimmunized donors.Techniques for the generation and use of antibody phage libraries arewell-known in the art. Once antibodies are identified, affinitymaturation strategies known in the art, including but not limited to,chain shuffling and site-directed mutagenesis, can be employed togenerate higher affinity human antibodies. In some embodiments, humanantibodies are produced in transgenic mice that contain humanimmunoglobulin loci. Upon immunization these mice are capable ofproducing the full repertoire of human antibodies in the absence ofendogenous immunoglobulin production.

The specific CDR sequences defined herein are generally based on acombination of Kabat and Chothia definitions. However, it is understoodthat reference to a heavy chain CDR or CDRs and/or a light chain CDR orCDRs of a specific antibody encompass all CDR definitions as known tothose of skill in the art.

Anti-BCMA antibodies or antigen-binding fragments provided hereininclude the followings clones: BCMA31The sequence features are describedbelow.

In some embodiments, anti-BCMA antibodies or antigen-binding fragmentsprovided herein (e.g., human BCMA) comprise one, two, three, four, five,and/or six CDRs of any one of the antibodies described herein. In someembodiments, anti-BCMA antibodies or antigen-binding fragments providedherein comprise a VL comprising one, two, and/or three, VL CDRs fromTable 1. In some embodiments, anti-BCMA antibodies or antigen-bindingfragments provided herein comprise a VH comprising one, two, and/orthree VH CDRs from Table 2. In some embodiments, anti-BCMA antibodies orantigen-binding fragments provided herein comprise one, two, and/orthree VL CDRs from Table 1 and one, two, and/or three VH CDRs from Table2.

TABLE 1 Amino acid sequences of light chain variableregion CDRs (VL CDRs) of anti-BCMA31 Antibody VL CDR1 VL CDR2 VL CDR3BCMA31 TGTSSDVGTYNYVS DVNQRPS SSYGGSNNLV (SEQ ID (SEQ ID (SEQ ID NO: 1)NO: 2) NO: 3)

TABLE 2 Amino acid sequences of heavy chain variableregion CDRs (VH CDRs) of anti-BCMA31 Antibody VH CDR1 VH CDR2 VH CDR3BCMA31 SYWMS NIKPDGSDK GATTYGS (SEQ ID GYYVDSVK (SEQ ID NO: 4) (SEQ IDNO: 6) NO: 5)

In some embodiments, an anti-BCMA antibody or antigen-binding fragmentthereof comprises a humanized antibody or antigen-binding fragment. Insome embodiments, an anti-BCMA antibody or antigen-binding fragmentthereof comprises a VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and/orVH CDR3 from an antibody or antigen-binding fragment described herein.In some embodiments, an anti-BCMA antibody or antigen-binding fragmentthereof comprises a variant of an anti-BCMA antibody or antigen-bindingfragment described herein. In some embodiments, a variant of ananti-BCMA antibody or antigen-binding fragment comprises one to 30 aminoacid substitutions, additions, and/or deletions in the anti-BCMAantibody or antigen-binding fragment. In some embodiments, a variant ofan anti-BCMA antibody or antigen-binding fragment comprises one to 25amino acid substitutions, additions, and/or deletions in the anti-BCMAantibody or antigen-binding fragment. In some embodiments, a variant ofan anti-BCMA antibody or antigen-binding fragment comprises one to 20substitutions, additions, and/or deletions in the anti-BCMA antibody orantigen-binding fragment. In some embodiments, a variant of an anti-BCMAantibody or antigen-binding fragment comprises one to 15 substitutions,additions, and/or deletions in the anti-BCMA antibody or antigen-bindingfragment. In some embodiments, a variant of an anti-BCMA antibody orantigen-binding fragment comprises one to 10 substitutions, additions,and/or deletions in the anti-BCMA antibody or antigen-binding fragment.In some embodiments, a variant of an anti-BCMA antibody orantigen-binding fragment comprises one to five conservative amino acidsubstitutions, additions, and/or deletions in the anti-BCMA antibody orantigen-binding fragment. In some embodiments, a variant of an anti-BCMAantibody or antigen-binding fragment comprises one to three amino acidsubstitutions, additions, and/or deletions in the anti-BCMA antibody orantigen-binding fragment. In some embodiments, the amino acidsubstitutions, additions, and/or deletions are conservative amino acidsubstitutions. In some embodiments, the conservative amino acidsubstitution(s) is in a CDR of the antibody or antigen-binding fragment.In some embodiments, the conservative amino acid substitution(s) is notin a CDR of the antibody or antigen-binding fragment. In someembodiments, the conservative amino acid substitution(s) is in aframework region of the antibody or antigen-binding fragment.

In some embodiments, provided herein are antibodies or antigen-bindingfragments thereof that specifically bind BCMA (e.g., human BCMA),comprising a light chain variable region (VL) comprising (1) a lightchain CDR1 (VL CDR1) having an amino acid sequence of SEQ ID NO:1; (2) alight chain CDR2 (VL CDR2) having an amino acid sequence consisting ofSEQ ID NO: 2; or (3) a light chain CDR3 (VL CDR3) having an amino acidsequence of SEQ ID NO: 3; or a variant thereof having up to about 3,about 5, about 8, about 10, about 12, or about 15 amino acidsubstitutions, additions, and/or deletions in the VL CDRs. In someembodiments, the variant has about 5 amino acid substitutions,additions, and/or deletions in the VL CDRs.

In some embodiments, provided herein are antibodies or antigen-bindingfragments thereof that specifically bind BCMA, comprising a VLcomprising (1) a VL CDR1 having an amino acid sequence of SEQ ID NO:1;(2) a VL CDR2 having an amino acid sequence consisting of SEQ ID NO:2;and (3) a VL CDR3 having an amino acid sequence of SEQ ID NO:3; or avariant thereof having up to about 3, about 5, about 8, about 10, about12, or about 15 amino acid substitutions, additions, and/or deletions inthe VL CDRs. In some embodiments, the variant has up to about 5 aminoacid substitutions, additions, and/or deletions in the VL CDRs.

In some embodiments, provided herein are antibodies or antigen-bindingfragments thereof that specifically bind BCMA (e.g., human BCMA)comprising a heavy chain variable region (VH) comprising (1) a heavychain CDR1 (VH CDR1) having an amino acid sequence of SEQ ID NO:4; (2) aheavy chain CDR2 (VH CDR2) having an amino acid sequence of SEQ ID NO:5;or (3) a heavy chain CDR3 (VH CDR3) having an amino acid sequence of SEQID NO:6; or a variant thereof having up to about 3, about 5, about 8,about 10, about 12, or about 15 amino acid substitutions, additions,and/or deletions in the VH CDRs. In some embodiments, the variant has upabout 5 amino acid substitutions, additions, and/or deletions in the VHCDRs.

In some embodiments, provided herein are antibodies or antigen-bindingfragments thereof that specifically bind BCMA (e.g., human BCMA)comprising a VH comprising (1) a VH CDR1 having an amino acid sequenceof SEQ ID NO:4; (2) a VH CDR2 having an amino acid sequence of SEQ IDNO:5; and (3) a VH CDR3 having an amino acid sequence of SEQ ID NO:6; ora variant thereof having up to about 3, about 5, about 8, about 10,about 12, or about 15 amino acid substitutions, additions, and/ordeletions in the VH CDRs. In some embodiments, the variant has up about5 amino acid substitutions, additions, and/or deletions in the VH CDRs.

In some embodiments, provided herein are antibodies or antigen-bindingfragments thereof that specifically bind BCMA (e.g., human BCMA),comprising (a) a VL comprising (1) a VL CDR1 having an amino acidsequence of SEQ ID NO:1; (2) a VL CDR2 having an amino acid sequence ofSEQ ID NO:2; and (3) a VL CDR3 having an amino acid sequence of SEQ IDNO:3; or a variant thereof having up to about 5 amino acidsubstitutions, additions, and/or deletions in the VL CDRs; and (b) a VHcomprising (1) a VH CDR1 having an amino acid sequence of SEQ ID NO:4;(2) a VH CDR2 having an amino acid sequence of SEQ ID NO:5; and (3) a VHCDR3 having an amino acid sequence of SEQ ID NO:6; or a variant thereofhaving up to about 5 amino acid substitutions, additions, and/ordeletions in the VH CDRs.

In some embodiments, provided herein are antibodies or antigen-bindingfragments thereof that specifically bind BCMA (e.g., human BCMA) havinga VL, wherein the VL comprises VL CDR1, CDR2 and CDR3 having the aminoacid sequences of SEQ ID NOs:1, 2, and 3, respectively, or a variantthereof having up to about 3, about 5, about 8, about 10, about 12, orabout 15 amino acid substitutions, additions, and/or deletions in the VLCDRs. In some embodiments, the variant has up about 5 amino acidsubstitutions, additions, and/or deletions in the VL CDRs.

In some embodiments, provided herein are antibodies or antigen-bindingfragments thereof that specifically bind BCMA (e.g., human BCMA) havinga VH, wherein the VH comprises VH CDR1, CDR2 and CDR3 having the aminoacid sequences of SEQ ID NOs:4, 5, and 6, respectively, or a variantthereof having up to about 3, about 5, about 8, about 10, about 12, orabout 15 amino acid substitutions, additions, and/or deletions in the VHCDRs. In some embodiments, the variant has up about 5 amino acidsubstitutions, additions, and/or deletions in the VH CDRs.

In some embodiments, provided herein are antibodies or antigen-bindingfragments thereof that specifically bind BCMA (e.g., human BCMA) havinga VL and a VH. In some embodiments, the VL and VH are connected by alinker. The linker can be a flexible linker or a rigid linker. In someembodiments, the linker has the amino acid sequence of (GGGGS)n, n=1, 2,3, 4, or 5. For example, the linker may have the amino acid sequence ofGGGGS (SEQ ID NO: 17). In some embodiments, the linker has the aminoacid sequence of (EAAAK)n, n=1, 2, 3, 4, or 5. For example, the linkermay have the amino acid sequence of EAAAK (SEQ ID NO: 18). In someembodiments, the linker has the amino acid sequence of (PA)nP, n=1, 2,3, 4, or 5. For example, the linker may have the amino acid sequence ofPAP (SEQ ID NO: 19). In some embodiments, the linker has the amino acidsequence of GGGGSGGGGSGGGGS (SEQ ID NO: 20).

In some embodiments, provided herein are antibodies or antigen-bindingfragments thereof that specifically bind BCMA (e.g., human BCMA) havinga VL and a VH, wherein (a) the VL comprises VL CDR1, CDR2 and CDR3having the amino acid sequences of SEQ ID NOs:1, 2, and 3, respectively,or a variant thereof having up to about 5 amino acid substitutions,additions, and/or deletions in the VL CDRs; and (b) the VH comprises VHCDR1, CDR2 and CDR3 having the amino acid sequences of SEQ ID NOs:4, 5,and 6, respectively; or a variant thereof having up to about 5 aminoacid substitutions, additions, and/or deletions in the VH CDRs.

In some embodiments, provided herein are antibodies or antigen-bindingfragments thereof that specifically bind BCMA (e.g., human BCMA) havinga VL and a VH, wherein the VL comprises VL CDR1, CDR2 and CDR3 and theVH comprises VH CDR1, CDR2 and CDR3, and wherein the VL CDR1, VL CDR2,VL CDR3, VH CDR1, VH CDR2 and VH CDR3 have the amino acid sequences ofSEQ ID NO:1, 2, 3, 4, 5, and 6, respectively, or a variant thereofhaving up to about 5 amino acid substitutions, additions, and/ordeletions in the CDRs.

In some embodiments, provided herein are antibodies or antigen-bindingfragments thereof that specifically bind BCMA (e.g., human BCMA) havinga VL, comprising (1) a VL CDR1 having the amino acid sequence of SEQ IDNO:1, (2) a VL CDR2 having the amino acid sequence of SEQ ID NO:2, or(3) a VL CDR3 having the amino acid sequence of SEQ ID NO:3. The VL canhave VL CDR1, VL CDR2, and VL CDR3 having the amino acid sequences ofSEQ ID NOs:1, 2, and 3, respectively. In some embodiments, providedherein are antibodies or antigen-binding fragments thereof thatspecifically bind BCMA having a VH, comprising (1) a VH CDR1 having theamino acid sequence of SEQ ID NO:4, (2) a VH CDR2 having the amino acidsequence of SEQ ID NO:5, or (3) a VH CDR3 having the amino acid sequenceof SEQ ID NO:6. The VH can have VH CDR1, VH CDR2, and VH CDR3 having theamino acid sequences of SEQ ID NOs:4, 5, and 6, respectively. In someembodiments, provided herein are antibodies or antigen-binding fragmentsthereof that specifically bind BCMA (e.g., human BCMA) comprising (a) aVL that comprises VL CDR1, VL CDR2, and VL CDR3 having the amino acidsequences of SEQ ID NOs:1, 2, and 3, respectively; and (b) a VH thatcomprises VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequencesof SEQ ID NOs:4, 5, and 6, respectively.

In some embodiments, provided herein are antibodies or antigen-bindingfragments thereof that specifically bind BCMA (e.g., human BCMA)comprising a VL having at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% sequence identity to an amino acidsequence of SEQ ID NO:7. In some embodiments, provided herein areantibodies or antigen-binding fragments thereof that specifically bindBCMA (e.g., human BCMA) comprising a VH having at least 85%, at least86%, at least 87%, at least 88%, at least 89%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, at least 99%, or 100% sequence identityto an amino acid sequence of SEQ ID NO: 8.

TABLE 3 Amino acid sequences of light chain variableregions (VL) and heavy chain variableregion (VH) of anti-BCMA31 antibodies Antibody VL VH BCMA31QSALTQPPSASGSP EVQLVESGGGLIQP GQSVTISCTGTSSD GGSLRLSCAASGFTVGTYNYVSWYQQHP FSSYWMSWVRQSPG GKAPKLMIYDVNQR KGLEWVANIKPDGSPSGVPDRFSGSKSG DKYYVDSVKGRFTI NTASLTVSGLQAED SRDNAKNSLDLQMNEADYYCSSYGGSNN SLRGEDTAIYYCAR LVFGGGTKVTVL GATTYGSWGQGT (SEQ ID NO: 7)LVTVSS (SEQ ID NO: 8)

In some embodiments, provided herein are antibodies or antigen-bindingfragments thereof that specifically bind BCMA (e.g., human BCMA)comprising: (a) a VL having at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% sequence identity sequence identity toan amino acid sequence of SEQ ID NO:7; and (b) a VH having at least 85%,at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100% sequenceidentity sequence identity to an amino acid sequence of SEQ ID NO:8.

In some embodiments, provided herein are antibodies or antigen-bindingfragments thereof that specifically bind BCMA (e.g., human BCMA)comprising a VL, wherein the VL has at least 80%, at least 85%, at least86%, at least 87%, at least 88%, at least 89%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, or at least 99% sequence identity toSEQ ID NO:7. In some embodiments, the anti-BCMA antibody orantigen-binding fragment thereof has a VL having at least 85% sequenceidentity to SEQ ID NO:7. In some embodiments, the anti-BCMA antibody orantigen-binding fragment thereof has a VL having at least 90% sequenceidentity to SEQ ID NO:7. In some embodiments, the anti-BCMA antibody orantigen-binding fragment thereof has a VL having at least 95% sequenceidentity to SEQ ID NO:7. In some embodiments, the anti-BCMA antibody orantigen-binding fragment thereof has a VL having at least 98% sequenceidentity to SEQ ID NO:7. In some embodiments, provided herein areantibodies or antigen-binding fragments thereof that specifically bindBCMA (e.g., human BCMA) comprising a VL having the amino acid sequenceof SEQ ID NO:7.

In some embodiments, provided herein are antibodies or antigen-bindingfragments thereof that specifically bind BCMA (e.g., human BCMA)comprising a VH, wherein the VH has at least 80%, at least 85%, at least86%, at least 87%, at least 88%, at least 89%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, or at least 99% sequence identity toSEQ ID NO:8. In some embodiments, the anti-BCMA antibody orantigen-binding fragment thereof has a VH having at least 85% sequenceidentity to SEQ ID NO:8. In some embodiments, the anti-BCMA antibody orantigen-binding fragment thereof has a VH having at least 90% sequenceidentity to SEQ ID NO:8. In some embodiments, the anti-BCMA antibody orantigen-binding fragment thereof has a VH having at least 95% sequenceidentity to SEQ ID NO:8. In some embodiments, the anti-BCMA antibody orantigen-binding fragment thereof has a VH having at least 98% sequenceidentity to SEQ ID NO:8. In some embodiments, provided herein areantibodies or antigen-binding fragments thereof that specifically bindBCMA (e.g., human BCMA) comprising a VH having the amino acid sequenceof SEQ ID NO:8.

The anti-BCMA antibodies or antigen-binding fragments thereof cancomprise a combination of any VL disclosed herein and any VH disclosedherein. In some embodiments, the VL and VH are connected by a linker.The linker can be a flexible linker or a rigid linker. In someembodiments, the linker has the amino acid sequence of (GGGGS)n, n=1, 2,3, 4, or 5. For example, the linker may have the amino acid sequence ofGGGGS (SEQ ID NO: 17). In some embodiments, the linker has the aminoacid sequence of (EAAAK)n, n=1, 2, 3, 4, or 5. For example, the linkermay have the amino acid sequence of EAAAK (SEQ ID NO: 18). In someembodiments, the linker has the amino acid sequence of (PA)nP, n=1, 2,3, 4, or 5. For example, the linker may have the amino acid sequence ofPAP (SEQ ID NO: 19). In some embodiments, the linker has the amino acidsequence of GGGGSGGGGSGGGGS (SEQ ID NO: 20).

In some embodiments, provided herein are antibodies or antigen-bindingfragments thereof that specifically bind BCMA (e.g., human BCMA)comprising a VL and a VH, wherein the VL and VH have the amino acidsequences of SEQ ID NO:7 and 8, respectively.

In some embodiments, provided herein are antibodies or antigen-bindingfragments thereof that specifically bind BCMA (e.g., human BCMA)comprising (a) a VL comprising VL CDRs from a VL having an amino acidsequence of SEQ ID NO:7; and/or (b) a VH comprising VH CDRs from a VHhaving an amino acid sequence of SEQ ID NO: 8.

In some embodiments, provided herein are antibodies or antigen-bindingfragments thereof that specifically bind BCMA (e.g., human BCMA)comprising a VL, wherein the VL comprises VL CDRs from a VL having theamino acid sequence of SEQ ID NO:7. In some embodiments, provided hereinare antibodies or antigen-binding fragments thereof that specificallybind BCMA (e.g., human BCMA) comprising a VH, wherein the VH comprisesVH CDRs from a VH having the amino acid sequence of SEQ ID NO: 8.

In some embodiments, provided herein are antibodies or antigen-bindingfragments thereof that specifically bind BCMA (e.g., human BCMA)comprising a VL and a VH, wherein the VL comprises VL CDR1, CDR2, andCDR3 from a VL having the amino acid sequence of SEQ ID NO:7, and the VHcomprises VH CDR1, CDR2, and CDR3 from a VH having the amino acidsequence of SEQ ID NO: 8.

In some embodiments, the anti-BCMA antibody or antigen-binding fragmentthereof provided herein is the scFv designated as BCMA31 (SEQ ID NO:11).In some embodiments, the anti-BCMA antibody or antigen-binding fragmentthereof provided herein has an amino acid sequence that is at leastabout 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about94%, about 95%, about 96%, about 97%, about 98%, or about 99% identicalto SEQ ID NO:11. In some embodiments, the anti-BCMA antibody orantigen-binding fragment thereof provided herein has a VL from BCMA31(SEQ ID NO:7). In some embodiments, the anti-BCMA antibody orantigen-binding fragment thereof provided herein has a VH from BCMA31(SEQ ID NO:8). The anti-BCMA antibody or antigen-binding fragmentthereof provided herein can have both a VL and a VH from BCMA31. In someembodiments, the anti-BCMA antibody or antigen-binding fragment thereofprovided herein has a VL that comprises VL CDRs from the VL from BCMA31(SEQ ID NO:7). In some embodiments, the anti-BCMA antibody orantigen-binding fragment thereof provided herein has a VH that comprisesVH CDRs from the VH from BCMA31 (SEQ ID NO:8). The anti-BCMA antibody orantigen-binding fragment thereof provided herein can have a VLcomprising VL CDRs and a VH comprising VH CDRs from the VL and VH ofBCMA31, respectively. In some embodiments, the anti-BCMA antibody orantigen-binding fragment thereof provided herein is a variant of BCMA31.The BCMA31 variant can have a VL that is a variant of the VL of BCMA31having up to about 5 amino acid substitutions, additions, and/ordeletions in SEQ ID NO:7. The BCMA31 variant can have a VH that is avariant of the VH of BCMA31 having up to about 5 amino acidsubstitutions, additions, and/or deletions in SEQ ID NO:8. The aminoacid substitutions, additions, and/or deletions can be in the VH CDRs orVL CDRs. In some embodiments, the amino acid substitutions, additions,and/or deletions are not in the CDRs. In some embodiments, the variantof BCMA31 has up to about 5 conservative amino acid substitutions. Insome embodiments, the variant of BCMA31 has up to 3 conservative aminoacid substitutions.

In some embodiments, provided herein are also antibodies orantigen-binding fragments that compete with the antibody orantigen-binding fragment provided above for binding to BCMA (e.g., humanBCMA). Antibodies that “compete with another antibody for binding to atarget” refer to antibodies that inhibit (partially or completely) thebinding of the other antibody to the target. Whether two antibodiescompete with each other for binding to a target, i.e., whether and towhat extent one antibody inhibits the binding of the other antibody to atarget, can be determined using known competition experiments, e.g.,BIACORE® surface plasmon resonance (SPR) analysis. In some embodiments,an anti-BCMA antibody or antigen-binding fragment competes with, andinhibits binding of another antibody or antigen-binding fragment to BCMAby at least 50%, 60%, 70%, 80%, 90% or 100%. Competition assays can beconducted as described, for example, in Ed Harlow and David Lane, ColdSpring Harb Protoc; 2006; doi: 10.H01/pdb.prot4277 or in Chapter 11 of“Using Antibodies” by Ed Harlow and David Lane, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., USA 1999.

In some embodiments, provided herein are antibodies or antigen-bindingfragments that compete with BCMA31 for binding to BCMA.

The present disclosure further contemplates additional variants andequivalents that are substantially homologous to the recombinant,monoclonal, chimeric, humanized, and human antibodies, or antibodyfragments thereof, described herein. In some embodiments, it isdesirable to improve the binding affinity of the antibody. In someembodiments, it is desirable to modulate biological properties of theantibody, including but not limited to, specificity, thermostability,expression level, effector function(s), glycosylation, immunogenicity,and/or solubility. Those skilled in the art will appreciate that aminoacid changes may alter post-translational processes of an antibody, suchas changing the number or position of glycosylation sites or alteringmembrane anchoring characteristics.

Variations can be a substitution, deletion, or insertion of one or morenucleotides encoding the antibody or polypeptide that results in achange in the amino acid sequence as compared with the native antibodyor polypeptide sequence. In some embodiments, amino acid substitutionsare the result of replacing one amino acid with another amino acidhaving similar structural and/or chemical properties, such as thereplacement of a leucine with a serine, e.g., conservative amino acidreplacements. Insertions or deletions can be in the range of about 1 to5 amino acids. In some embodiments, the substitution, deletion, orinsertion includes less than 25 amino acid substitutions, less than 20amino acid substitutions, less than 15 amino acid substitutions, lessthan 10 amino acid substitutions, less than 5 amino acid substitutions,less than 4 amino acid substitutions, less than 3 amino acidsubstitutions, or less than 2 amino acid substitutions relative to theparent molecule. In some embodiments, variations in the amino acidsequence that are biologically useful and/or relevant can be determinedby systematically making insertions, deletions, or substitutions in thesequence and testing the resulting variant proteins for activity ascompared to the parent protein.

It is known in the art that the constant region(s) of an antibodymediates several effector functions and these effector functions canvary depending on the isotype of the antibody. For example, binding ofthe Cl component of complement to the Fc region of IgG or IgM antibodies(bound to antigen) activates the complement system. Activation ofcomplement is important in the opsonization and lysis of cell pathogens.The activation of complement also stimulates the inflammatory responseand can be involved in autoimmune hypersensitivity. In addition, the Fcregion of an antibody can bind a cell expressing a Fc receptor (FcR).There are a number of Fc receptors which are specific for differentclasses of antibody, including IgG (gamma receptors), IgE (epsilonreceptors), IgA (alpha receptors) and IgM (mu receptors). Binding ofantibody to Fc receptors on cell surfaces triggers a number of importantand diverse biological responses including engulfment and destruction ofantibody-coated particles, clearance of immune complexes, lysis ofantibody-coated target cells by killer cells (called antibody-dependentcell cytotoxicity or ADCC), release of inflammatory mediators, placentaltransfer, and control of immunoglobulin production. In some embodiments,anti-BCMA antibody or antigen-binding fragment described herein compriseat least one constant region of a human IgA antibody. In someembodiments, anti-BCMA antibody or antigen-binding fragment describedherein comprise at least one constant region of a human IgD antibody. Insome embodiments, anti-BCMA antibody or antigen-binding fragmentdescribed herein comprise at least one constant region of a human IgEantibody. In some embodiments, anti-BCMA antibody or antigen-bindingfragment described herein comprise at least one constant region of ahuman IgG antibody. In some embodiments, anti-BCMA antibody orantigen-binding fragment described herein comprise at least one constantregion of a human IgM antibody. In some embodiments, anti-BCMA antibodyor antigen-binding fragment described herein comprise at least oneconstant region of a human IgG1 antibody. In some embodiments, anti-BCMAantibody or antigen-binding fragment described herein comprise at leastone constant region of a human IgG2 antibody. In some embodiments,anti-BCMA antibody or antigen-binding fragment described herein compriseat least one constant region of a human IgG3 antibody. In someembodiments, anti-BCMA antibody or antigen-binding fragment describedherein comprise at least one constant region of a human IgG4 antibody.

In some embodiments, at least one or more of the constant regions hasbeen modified or deleted in the anti-BCMA antibody or antigen-bindingfragment described herein. In some embodiments, the antibodies comprisemodifications to one or more of the three heavy chain constant regions(CH1, CH2 or CH3) and/or to the light chain constant region (CL). Insome embodiments, the heavy chain constant region of the modifiedantibodies comprises at least one human constant region. In someembodiments, the heavy chain constant region of the modified antibodiescomprises more than one human constant region. In some embodiments,modifications to the constant region comprise additions, deletions, orsubstitutions of one or more amino acids in one or more regions. In someembodiments, one or more regions are partially or entirely deleted fromthe constant regions of the modified antibodies. In some embodiments,the entire CH2 domain has been removed from an antibody (ΔCH2constructs). In some embodiments, a deleted constant region is replacedby a short amino acid spacer that provides some of the molecularflexibility typically imparted by the absent constant region. In someembodiments, a modified antibody comprises a CH3 domain directly fusedto the hinge region of the antibody. In some embodiments, a modifiedantibody comprises a peptide spacer inserted between the hinge regionand modified CH2 and/or CH3 domains.

In some embodiments, an anti-BCMA antibody or antigen-binding fragmentcomprises a Fc region. In some embodiments, the Fc region is fused via ahinge. The hinge can be an IgG1 hinge, an IgG2 hinge, or an IgG3 hinge.The amino acid sequences of the Fc region of human IgG1, IgG2, IgG3, andIgG4 are known to those of ordinary skill in the art. In some cases, Fcregions with amino acid variations have been identified in nativeantibodies. In some embodiments, the modified antibodies (e.g., modifiedFc region) provide for altered effector functions that, in turn, affectthe biological profile of the antibody. For example, in someembodiments, the deletion or inactivation (through point mutations orother means) of a constant region reduces Fc receptor binding of themodified antibody as it circulates. In some embodiments, the constantregion modifications reduce the immunogenicity of the antibody. In someembodiments, the constant region modifications increase the serumhalf-life of the antibody. In some embodiments, the constant regionmodifications reduce the serum half-life of the antibody. In someembodiments, the constant region modifications decrease or remove ADCCand/or complement dependent cytotoxicity (CDC) of the antibody. In someembodiments, specific amino acid substitutions in a human IgG1 Fc regionwith corresponding IgG2 or IgG4 residues reduce effector functions(e.g., ADCC and CDC) in the modified antibody. In some embodiments, anantibody does not have one or more effector functions (e.g.,“effectorless” antibodies). In some embodiments, the antibody has noADCC activity and/or no CDC activity. In some embodiments, the antibodydoes not bind an Fc receptor and/or complement factors. In someembodiments, the antibody has no effector function(s). In someembodiments, the constant region modifications increase or enhance ADCCand/or CDC of the antibody. In some embodiments, the constant region ismodified to eliminate disulfide linkages or oligosaccharide moieties. Insome embodiments, the constant region is modified to add/substitute oneor more amino acids to provide one or more cytotoxin, oligosaccharide,or carbohydrate attachment sites. In some embodiments, an anti-BCMAantibody or antigen-binding fragment comprises a variant Fc region thatis engineered with substitutions at specific amino acid positions ascompared to a native Fc region. In some embodiments, an anti-BCMAantibody or antigen-binding fragment described herein comprises an IgG1heavy chain constant region that comprises one or more amino acidsubstitutions selected from the group consisting of K214R, L234A, L235E,G237A, D356E, and L358M, per EU numbering. In some embodiments, the IgG1heavy chain constant region comprises one or more amino acidsubstitutions selected from the group consisting of K214R, L234A, L235E,G237A, A330S, P331S, D356E, and L358M, per EU numbering. In someembodiments, the IgG1 heavy chain constant region comprises one or moreamino acid substitutions selected from the group consisting of K214R,C226S, C229S, and P238S, per EU numbering. In some embodiments, the IgG1heavy chain constant region comprises one or more amino acidsubstitutions selected from the group consisting of K214R, D356E, andL358M, per EU numbering. In some embodiments, the IgG1 heavy chainconstant region comprises one or more amino acid substitutions selectedfrom the group consisting of S131C, K133R, G137E, G138S, Q196K, I199T,N203D, K214R, C226S, C229S, and P238S, per EU numbering.

In some embodiments, variants can include addition of amino acidresidues at the amino- and/or carboxyl-terminal end of the antibody orpolypeptide. The length of additional amino acids residues can rangefrom one residue to a hundred or more residues. In some embodiments, avariant comprises an N-terminal methionyl residue. In some embodiments,the variant comprises an additional polypeptide/protein (e.g., Fcregion) to create a fusion protein. In some embodiments, a variant isengineered to be detectable and may comprise a detectable label and/orprotein (e.g., a fluorescent tag or an enzyme).

The variant antibodies or antigen-binding fragments described herein canbe generated using methods known in the art, including but not limitedto, site-directed mutagenesis, alanine scanning mutagenesis, and PCRmutagenesis.

In some embodiments, a variant of an anti-BCMA antibody orantigen-binding fragment disclosed herein can retain the ability to bindBCMA to a similar extent, the same extent, or to a higher extent, as theparent antibody or antigen-binding fragment. In some embodiments, thevariant can be at least about 80%, about 85%, about 90%, about 91%,about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about98%, about 99% or more identical in amino acid sequence to the parentantibody or antigen-binding fragment. In certain embodiments, a variantof an anti-BCMA antibody or antigen-binding fragment comprises the aminoacid sequence of the parent anti-BCMA antibody or antigen-bindingfragment with one or more conservative amino acid substitution.Conservative amino acid substitutions are known in the art and includeamino acid substitutions in which one amino acid having certain physicaland/or chemical properties is exchanged for another amino acid that hasthe same or similar chemical or physical properties.

In some embodiments, a variant of an anti-BCMA antibody orantigen-binding fragment comprises the amino acid sequence of the parentantibody or antigen-binding fragment with one or more non-conservativeamino acid substitutions. In some embodiments, a variant of an anti-BCMAantibody or antigen-binding fragment comprises the amino acid sequenceof the parent binding antibody or antigen-binding fragment with one ormore non-conservative amino acid substitution, wherein the one or morenon-conservative amino acid substitutions do not interfere with orinhibit one or more biological activities of the variant (e.g., BCMAbinding). In certain embodiments, the one or more conservative aminoacid substitutions and/or the one or more non-conservative amino acidsubstitutions can enhance a biological activity of the variant, suchthat the biological activity of the functional variant is increased ascompared to the parent binding moiety.

In some embodiments, the variant can have 1, 2, 3, 4, or 5 amino acidsubstitutions in the CDRs (e.g., VH CDR1, VH CDR2, VH CDR3, VL CDR1, VLCDR2 and VL CDR3) of the binding moiety.

In some embodiments, anti-BCMA antibodies or antigen-binding fragmentsdescribed herein are chemically modified naturally or by intervention.In some embodiments, the anti-BCMA antibodies or antigen-bindingfragments have been chemically modified by glycosylation, acetylation,pegylation, phosphorylation, amidation, derivatization by knownprotecting/blocking groups, proteolytic cleavage, and/or linkage to acellular ligand or other protein. Any of numerous chemical modificationscan be carried out by known techniques. The anti-BCMA antibodies orantigen-binding fragments can comprise one or more analogs of an aminoacid (including, for example, unnatural amino acids), as well as othermodifications known in the art.

In some embodiments, an anti-BCMA antibody or antigen-binding fragment(e.g., an antibody) binds BCMA (e.g., human BCMA) with a dissociationconstant (K_(D)) of about 1 μM or less, about 100 nM or less, about 40nM or less, about 20 nM or less, about 10 nM or less, about 1 nM orless, about 0.1 nM or less, 50 pM or less, 10 pM or less, or 1 pM orless. In some embodiments, an anti-BCMA antibody or antigen-bindingfragment binds BCMA (e.g., human BCMA) with a K_(D) of about 20 nM orless. In some embodiments, an anti-BCMA antibody or antigen-bindingfragment binds BCMA (e.g., human BCMA) with a K_(D) of about 10 nM orless. In some embodiments, an anti-BCMA antibody or antigen-bindingfragment binds BCMA (e.g., human BCMA) with a K_(D) of about 1 nM orless. In some embodiments, an anti-BCMA antibody or antigen-bindingfragment binds BCMA (e.g., human BCMA) with a K_(D) of about 0.5 nM orless. In some embodiments, an anti-BCMA antibody or antigen-bindingfragment binds BCMA (e.g., human BCMA) with a K_(D) of about 0.1 nM orless. In some embodiments, an anti-BCMA antibody or antigen-bindingfragment binds BCMA (e.g., human BCMA) with a K_(D) of about 50 pM orless. In some embodiments, an anti-BCMA antibody or antigen-bindingfragment binds BCMA (e.g., human BCMA) with a K_(D) of about 25 pM orless. In some embodiments, an anti-BCMA antibody or antigen-bindingfragment binds BCMA (e.g., human BCMA) with a K_(D) of about 10 pM orless. In some embodiments, an anti-BCMA antibody or antigen-bindingfragment binds BCMA (e.g., human BCMA) with a K_(D) of about 1 pM orless. In some embodiments, the dissociation constant of the bindingagent (e.g., an antibody) for BCMA is the dissociation constantdetermined using a BCMA protein immobilized on a Biacore chip and thebinding agent flowed over the chip. In some embodiments, thedissociation constant of the binding agent (e.g., an antibody) for BCMAis the dissociation constant determined using the binding agent capturedby an anti-human IgG antibody on a Biacore chip and soluble BCMA flowedover the chip.

The anti-BCMA antibodies or antigen-binding fragments of the presentdisclosure can be analyzed for their physical, chemical and/orbiological properties by various methods known in the art. In someembodiments, an anti-BCMA antibody is tested for its ability to bindBCMA (e.g., human BCMA). Binding assays include, but are not limited to,SPR (e.g., Biacore), ELISA, and FACS. In addition, antibodies can beevaluated for solubility, stability, thermostability, viscosity,expression levels, expression quality, and/or purification efficiency.

Epitope mapping is a method of identifying the binding site, region, orepitope on a target protein where an antibody binds. A variety ofmethods are known in the art for mapping epitopes on target proteins.These methods include mutagenesis, including but not limited to, shotgunmutagenesis, site-directed mutagenesis, and alanine scanning; domain orfragment scanning; peptide scanning (e.g., Pepscan technology); displaymethods (e.g., phage display, microbial display, and ribosome/mRNAdisplay); methods involving proteolysis and mass spectroscopy; andstructural determination (e.g., X-ray crystallography and NMR). In someembodiments, anti-BCMA antibodies or antigen-binding fragments describedherein are characterized by assays including, but not limited to,N-terminal sequencing, amino acid analysis, HPLC, mass spectrometry, ionexchange chromatography, and papain digestion.

In some embodiments, an anti-BCMA antibody or antigen-binding fragmentis conjugated to a cytotoxic agent or moiety. In some embodiments, ananti-BCMA antibody or antigen-binding fragment is conjugated to acytotoxic agent to form an ADC (antibody-drug conjugate). In someembodiments, the cytotoxic moiety is a chemotherapeutic agent including,but not limited to, methotrexate, adriamycin/doxorubicin, melphalan,mitomycin C, chlorambucil, duocarmycin, daunorubicin,pyrrolobenzodiazepines (PBDs), or other intercalating agents. In someembodiments, the cytotoxic moiety is a microtubule inhibitor including,but not limited to, auristatins, maytansinoids (e.g., DM1 and DM4), andtubulysins. In some embodiments, the cytotoxic moiety is anenzymatically active toxin of bacterial, fungal, plant, or animalorigin, or fragments thereof, including, but not limited to, diphtheriaA chain, non-binding active fragments of diphtheria toxin, exotoxin Achain, ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin,Aleurites fordii proteins, dianthin proteins, Phytolaca americanaproteins (PAPI, PAPII, and PAP-S), Momordica charantia inhibitor,curcin, crotin, Sapaonaria officinalis inhibitor, gelonin, mitogellin,restrictocin, phenomycin, enomycin, and the tricothecenes. In someembodiments, an antibody is conjugated to one or more small moleculetoxins, such as calicheamicins, maytansinoids, trichothenes, and CC1065.

In some embodiments, an anti-BCMA antibody or antigen-binding fragmentdescribed herein is conjugated to a detectable substance or moleculethat allows the agent to be used for diagnosis and/or detection. Adetectable substance can include, but is not limited to, enzymes, suchas horseradish peroxidase, alkaline phosphatase, beta-galactosidase, andacetylcholinesterase; prosthetic groups, such as biotin and flavine(s);fluorescent materials, such as, umbelliferone, fluorescein, fluoresceinisothiocyanate (FITC), rhodamine, tetramethylrhodamine isothiocyanate(TRITC), dichlorotriazinylamine fluorescein, dansyl chloride, cyanine(Cy3), and phycoerythrin; bioluminescent materials, such as luciferase;radioactive materials, such as ²¹²Bi, ¹⁴C, ⁵⁷Co, ⁵¹Cr, ⁶⁷Cu, ¹⁸F, ⁶⁸Ga,⁶⁷Ga, ¹⁵³Gd, ¹⁵⁹Gd, ⁶⁸Ge, ³H, ¹⁶⁶Ho, ¹³¹I, ¹²⁵I, ¹²³I, ¹²¹I, ¹¹⁵In,¹¹³In, ¹¹²In, ¹¹¹In, ¹⁴⁰La, ¹⁷⁷Lu, ⁵⁴Mn, ⁹⁹Mo, ³²P, ¹⁰³Pd, ¹⁴⁹Pm, ¹⁴²Pr,¹⁸⁶Re, ¹⁸⁸Re, ¹⁰⁵Rh, ⁹⁷Ru, ³⁵S, ⁴⁷Sc, ⁷⁵Se, ¹⁵³Sm, ¹¹³Sn, ¹¹⁷Sn, ⁸⁵Sr,^(99m)Tc, ²⁰¹Ti, ¹³³Xe, ⁹⁰Y, ⁶⁹Yb, ¹⁷⁵Yb, ⁶⁵Zn; positron emittingmetals; and magnetic metal ions positron emitting metals; and magneticmetal ions.

An anti-BCMA antibody or antigen-binding fragment described herein canbe attached to a solid support. Such solid supports include, but are notlimited to, glass, cellulose, polyacrylamide, nylon, polystyrene,polyvinyl chloride, or polypropylene. In some embodiments, animmobilized anti-BCMA antibody or antigen-binding fragment is used in animmunoassay. In some embodiments, an immobilized anti-BCMA antibody orantigen-binding fragment is used in purification of the target antigen(e.g., human BCMA).

5.3 CARs, TCRs and Genetically Engineered Immune Effector Cells

The anti-BCMA antibodies or antigen-binding fragments described hereincan be used as part of a chimeric antigen receptor (CAR) or a T-CellReceptor (TCR) that can be expressed in an immune effector cell forcancer treatment. As such, provided herein are also CARs and TCRs thatspecifically bind BCMA, immune effector cells that express such CARs orTCRs, and the uses of such cells.

5.3.1 TCRs

Provided herein are T cell receptors (TCRs) that specifically bind BCMA(“BCMA TCR”). TCRs are antigen-specific molecules that are responsiblefor recognizing antigenic peptides presented in the context of a productof the MEC on the surface of APCs or any nucleated cells. This systemendows T cells, via their TCRs, with the potential ability to recognizethe entire array of intracellular antigens expressed by a cell(including virus proteins) that are processed into short peptides, boundto an intracellular MEC molecule, and delivered to the surface as apeptide-MEC complex. This system allows foreign protein (e.g., mutatedcancer antigen or virus protein) or aberrantly expressed protein toserve a target for T cells (e.g., Davis and Bjorkman (1988) Nature, 334,395-402; Davis et al. (1998) Annu Rev Immunol, 16, 523-544).

The interaction of a TCR and a peptide-MEC complex can drive the T cellinto various states of activation, depending on the affinity (ordissociation rate) of binding. The TCR recognition process allows a Tcell to discriminate between a normal, healthy cell and, for example,one that has become transformed via a virus or malignancy, by providinga diverse repertoire of TCRs, wherein there is a high probability thatone or more TCRs will be present with a binding affinity for the foreignpeptide bound to an MEC molecule that is above the threshold forstimulating T cell activity (Manning and Kranz (1999) Immunology Today,20, 417-422).

Wild type TCRs isolated from either human or mouse T cell clones thatwere identified by in vitro culturing have been shown to have relativelylow binding affinities (K_(D)=1-300 μM) (Davis et al. (1998) Annu RevImmunol, 16, 523-544). This is partly because that T cells that developin the thymus are negatively selected (tolerance induction) onself-peptide-MHC ligands, such that T cells with too high of an affinityare deleted (Starr et al. (2003) Annu Rev Immunol, 21, 139-76). Tocompensate for these relatively low affinities, T cells have evolved aco-receptor system in which the cell surface molecules CD4 and CD8 bindto the MEC molecules (class II and class I, respectively) and synergizewith the TCR in mediating signaling activity. CD8 is particularlyeffective in this process, allowing TCRs with very low affinity (e.g.,K_(D)=300 μM) to mediate potent antigen-specific activity.

Directed evolution can be used to generate TCRs with higher affinity fora specific peptide-MEC complex. Methods that can be used include yeastdisplay (Holler et al. (2003) Nat Immunol, 4, 55-62; Holler et al.(2000) Proc Natl Acad Sci USA, 97, 5387-92), phage display (Li et al.(2005) Nat Biotechnol, 23, 349-54), and T cell display (Chervin et al.(2008) J Immunol Methods, 339, 175-84). All three approaches involveengineering, or modifying, a TCR that exhibits the normal, low affinityof the wild-type TCR, to increase the affinity for the cognatepeptide-MEC complex (the original antigen that the T cells were specificfor).

As such, in some embodiments, provided herein are TCRs comprising ananti-BCMA antibody or antigen-binding fragment described herein. Theanti-BCMA antibody or antigen-binding fragment can be any anti-BCMAantibody or antigen-binding fragment described herein. For illustrativepurposes, in some embodiments, the TCRs provided herein can comprise ananti-BCMA antibody or antigen-binding fragment having a VL and a VH,wherein the VL comprises VL CDR1, CDR2 and CDR3 and the VH comprises VHCDR1, CDR2 and CDR3, and wherein the VL CDR1, VL CDR2, VL CDR3, VH CDR1,VH CDR2 and VH CDR3 have the amino acid sequences of SEQ ID NO:1, 2, 3,4, 5, and 6.

In some embodiments, the TCRs provided herein can comprise an anti-BCMAantibody or antigen-binding fragment that is the scFv designated asBCMA31.

In some embodiments, the TCRs provided herein comprise an alpha (α)chain and a beta (β) chain. The constant region of TCR α chain and 13chain are encoded by TRAC and TRBC, respectively. A human TRAC can havean amino acid sequence corresponding to UniProtKB/Swiss-Prot No.:P01848.2 (Accession: P01848.2 GI: 1431906459). A human TRBC can have anamino acid sequence corresponding to the GenBank sequence ALC78509.1(Accession: ALC78509.1 GI: 924924895). In some embodiments, the TCRsprovided herein comprise a TCR α chain comprising an anti-BCMA antibodyor antigen-binding fragment provided herein. In some embodiments, theTCRs provided herein comprise a TCR β chain comprising an anti-BCMAantibody or antigen-binding fragment provided herein. In someembodiments, the TCR comprises a gamma chain (γ) and a delta (δ) chain.The constant region of TCR γ chain and δ chain are encoded by encoded byTRGC and TRDC, respectively. A human TRGC can have an amino acidsequence corresponding to UniProtKB/Swiss-Prot: P0CF51.1 (Accession:P0CF51.1 GI: 294863156), or an amino acid sequence corresponding toUniProtKB/Swiss-Prot: P03986.2 (Accession: P03986.2 GI: 1531253869). Ahuman TRDC can have an amino acid sequence corresponding to theUniProtKB/Swiss-Prot: B7Z8K6.2 (Accession: B7Z8K6.2 GI: 294863191). Insome embodiments, the TCRs provided herein comprise a TCR γ chaincomprising an anti-BCMA antibody or antigen-binding fragment providedherein. In some embodiments, the TCRs provided herein comprise a TCR δchain comprising an anti-BCMA antibody or antigen-binding fragmentprovided herein.

5.3.2 CARs

CARs are engineered receptors that provide both antigen binding andimmune effector cell activation functions. CARs can be used to graft thespecificity of an antibody, such as a monoclonal antibody, onto animmune effector cell such as a T cell, a NK cell, or a macrophage. CARscan retarget immune effector cells (e.g., T cells) to tumor surfaceantigens in HLA-independent manner (Sadelain et al., Nat. Rev. Cancer.3(1):35-45 (2003); Sadelain et al., Cancer Discovery 3(4):388-398(2013); Rafiq and Brentjens (2016). Nat Rev Clin Oncol 13(6): 370-383).The typical structure of a CAR molecule includes an extracellularantigen-binding domain (e.g., scFv), a transmembrane domain (TM) and anintracellular signaling domain. The extracellular antigen-binding domainof a CAR is usually derived from a monoclonal antibody (mAb) or fromreceptors or their ligands. Antigen binding by the CARs triggersphosphorylation of immunoreceptor tyrosine-based activation motifs(ITAMs) in the intracellular domain, initiating a signaling cascaderequired for cytolysis induction, cytokine secretion, and proliferation.CAR-expressing T cells (“CART”s) can be classified into threegenerations according to the presence of intracellular co-stimulatorysignals.

In some embodiments, provided herein are CARs that specifically bindsBCMA (“BCMA CAR”). In some embodiments, the CAR can be a “firstgeneration,” “second generation” or “third generation” CAR (see, forexample, Sadelain et al., Cancer Discov. 3(4):388-398 (2013); Jensen etal, Immunol. Rev. 257:127-133 (2014); Sharpe et al, Dis. Model Mech.8(4):337-350 (2015); June et al (2018), Science 359(6382): 1361-1365).

“First generation” CARs are typically composed of an extracellularantigen binding domain, for example, a single-chain variable fragment(scFv), fused to a transmembrane domain, which is fused to acytoplasmic/intracellular domain of the T cell receptor chain. “Firstgeneration” CARs typically have the intracellular domain from theCD3ζ-chain, which is the primary transmitter of signals from endogenousT cell receptors (TCRs). “First generation” CARs can provide de novoantigen recognition and cause activation of both CD4⁺ and CD8⁺ T cellsthrough their CD3ζ chain signaling domain in a single fusion molecule,independent of HLA-mediated antigen presentation. “Second-generation”CARs comprise a cancer antigen-binding domain fused to an intracellularsignaling domain capable of activating immune effector cells such as Tcells and a co-stimulatory domain designed to augment immune effectorcell, such as T cell, potency and persistence (Sadelain et al., CancerDiscov. 3:388-398 (2013)). CAR design can therefore combine antigenrecognition with signal transduction, two functions that arephysiologically borne by two separate complexes, the TCR heterodimer andthe CD3 complex. “Second generation” CARs include an intracellulardomain from various co-stimulatory receptors, for example, CD28, 4-1BB,ICOS, OX40, and the like, in the cytoplasmic tail of the CAR to provideadditional signals to the cell. “Second generation” CARs provide bothco-stimulation, for example, by CD28 or 4-1BB domains, and activation,for example, by a CD3 signaling domain. Studies have indicated that“Second Generation” CARs can improve the anti-tumor activity of T cells.In 2017, FDA approved two anti-CD19 CART cell products for the treatmentof relapsed B-cell precursor acute lymphoblastic leukemia (B-ALL) andB-cell Non-Hodgkin Lymphoma. “Third generation” CARs provide multipleco-stimulation, for example, by comprising both CD28 and 4-1BB domains,and activation, for example, by comprising a CD3ζ activation domain.

As such, provided herein are CARs that specifically binds BCMA,comprising, from N-terminus to C-terminus: (a) a BCMA binding domaincomprising an anti-BCMA antibody or antigen-binding fragment providedherein, (b) a transmembrane domain, and (c) a cytoplasmic domain. Theanti-BCMA antibody or antigen-binding fragment can be any anti-BCMAantibody or antigen-binding fragment described herein. For illustrativepurposes, in some embodiments, the CARs provided herein can comprise ananti-BCMA antibody or antigen-binding fragment having a VL and a VH,wherein the VL comprises VL CDR1, CDR2 and CDR3 and the VH comprises VHCDR1, CDR2 and CDR3, and wherein the VL CDR1, VL CDR2, VL CDR3, VH CDR1,VH CDR2 and VH CDR3 have the amino acid sequences of SEQ ID NO:1, 2, 3,4, 5, and 6, respectively.

In some embodiments, the CARs provided herein can comprise an anti-BCMAantibody or antigen-binding fragment that is the scFv designated asBCMA31.

In some embodiments, the transmembrane domain of the CARs providedherein comprises a hydrophobic alpha helix that spans at least a portionof the membrane. Different transmembrane domains result in differentreceptor stability. After antigen recognition, receptors cluster and asignal is transmitted to the cell. In some embodiments, thetransmembrane domain of the CAR provided herein can be derived from aprotein or polypeptide that is naturally expressed in an immune effectorcell. A transmembrane domain derived from a protein or polypeptide meansthat the transmembrane domain comprises the entire transmembrane regionof the protein or polypeptide, or a fragment thereof. In someembodiments, the CAR provided herein can have a transmembrane domainderived from CD8, CD28, CD3ζ, CD4, 4-1BB, OX40, ICOS, CTLA-4, PD-1,LAG-3, 2B4, BTLA, T-cell receptor (TCR) α chain, TCR β chain, or TCR ζchain, CDRε, CD45, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80,CD86, CD134, CD154, or other polypeptides expressed in the immuneeffector cell. In some embodiments, the transmembrane domain of CARsprovided herein comprises the transmembrane region of CD8, CD28, CD3ζ,CD4, 4-1BB, OX40, ICOS, CTLA-4, PD-1, LAG-3, 2B4, BTLA, T-cell receptor(TCR) α chain, TCR β chain, or TCR ζ chain, CD3ε, CD45, CD5, CD8, CD9,CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD154, or otherpolypeptides expressed in the immune effector cell.

In some embodiments, the transmembrane domain of CARs provided herein isderived from CD8. In some embodiments, the transmembrane domaincomprises the transmembrane region of CD8. In some embodiments, thetransmembrane domain is derived from CD28. In some embodiments, thetransmembrane domain comprises the transmembrane region of CD28. In someembodiments, the transmembrane domain is derived from CD3ζ. In someembodiments, the transmembrane domain comprises the transmembrane regionof CD3ζ. In some embodiments, the transmembrane domain is derived fromCD4. In some embodiments, the transmembrane domain comprises thetransmembrane region of CD4. In some embodiments, the transmembranedomain is derived from 4-1BB. In some embodiments, the transmembranedomain comprises the transmembrane region of 4-1BB. In some embodiments,the transmembrane domain is derived from OX40. In some embodiments, thetransmembrane domain comprises the transmembrane region of OX40. In someembodiments, the transmembrane domain is derived from ICOS. In someembodiments, the transmembrane domain comprises the transmembrane regionof ICOS. In some embodiments, the transmembrane domain is derived fromCTLA-4. In some embodiments, the transmembrane domain comprises thetransmembrane region of CTLA-4. In some embodiments, the transmembranedomain is derived from PD-1. In some embodiments, the transmembranedomain comprises the transmembrane region of PD-1. In some embodiments,the transmembrane domain is derived from LAG-3. In some embodiments, thetransmembrane domain comprises the transmembrane region of LAG-3. Insome embodiments, the transmembrane domain is derived from 2B4. In someembodiments, the transmembrane domain comprises the transmembrane regionof 2B4. In some embodiments, the transmembrane domain is derived fromBTLA. In some embodiments, the transmembrane domain comprises thetransmembrane region of BTLA. In some embodiments, the transmembranedomain is derived from TCR α chain. In some embodiments, thetransmembrane domain comprises the transmembrane region of TCR α chain.In some embodiments, the transmembrane domain is derived from TCR βchain. In some embodiments, the transmembrane domain comprises thetransmembrane region of TCR chain. In some embodiments, thetransmembrane domain is derived from TCR ζ chain. In some embodiments,the transmembrane domain comprises the transmembrane region of TCR ζchain. In some embodiments, the transmembrane domain is derived fromCD3ε. In some embodiments, the transmembrane domain comprises thetransmembrane region of CD3ε. In some embodiments, the transmembranedomain is derived from CD45. In some embodiments, the transmembranedomain comprises the transmembrane region of CD45. In some embodiments,the transmembrane domain is derived from CD5. In some embodiments, thetransmembrane domain comprises the transmembrane region of CD5. In someembodiments, the transmembrane domain is derived from CD8. In someembodiments, the transmembrane domain comprises the transmembrane regionof CD8. In some embodiments, the transmembrane domain is derived fromCD9. In some embodiments, the transmembrane domain comprises thetransmembrane region of CD9. In some embodiments, the transmembranedomain is derived from CD16. In some embodiments, the transmembranedomain comprises the transmembrane region of CD16. In some embodiments,the transmembrane domain is derived from CD22. In some embodiments, thetransmembrane domain comprises the transmembrane region of CD22. In someembodiments, the transmembrane domain is derived from CD33. In someembodiments, the transmembrane domain comprises the transmembrane regionof CD33. In some embodiments, the transmembrane domain is derived fromCD37. In some embodiments, the transmembrane domain comprises thetransmembrane region of CD37. In some embodiments, the transmembranedomain is derived from CD64. In some embodiments, the transmembranedomain comprises the transmembrane region of CD64. In some embodiments,the transmembrane domain is derived from CD80. In some embodiments, thetransmembrane domain comprises the transmembrane region of CD80. In someembodiments, the transmembrane domain is derived from CD86. In someembodiments, the transmembrane domain comprises the transmembrane regionof CD86. In some embodiments, the transmembrane domain is derived fromCD134. In some embodiments, the transmembrane domain comprises thetransmembrane region of CD134. In some embodiments, the transmembranedomain is derived from CD154. In some embodiments, the transmembranedomain comprises the transmembrane region of CD154. Exemplarytransmembrane domains are described below in more detail.

In some embodiments, the transmembrane domain can be synthetic, in whichcase it comprises predominantly hydrophobic residues such as leucine andvaline. Optionally, the transmembrane domain can be derived from apolypeptide that is not naturally expressed in the immune effector cell,so long as the transmembrane domain can function in transducing signalfrom antigen bound to the CAR to the intracellular signaling and/orco-stimulatory domains. In some embodiments, the transmembrane domaincan comprise a triplet of phenylalanine, tryptophan and valine at eachend. Optionally, a short oligo- or polypeptide linker, preferablybetween 2 and 10 amino acids in length can form the linkage between thetransmembrane domain and the cytoplasmic signaling domain of the CAR. Aglycine-serine doublet provides a particularly suitable linker.

Cytoplasmic domains of CARs provided herein can contain a signalingdomain that functions in the immune effector cell expressing the CAR.Such a signaling domain can be, for example, derived from CD3ζ, Fcreceptor γ, FcγRIIa, FcRβ (Fcε1b), CD3γ, CD3δ, CD3ε, CD79a, CD79b,DAP10, or DAP12. A signaling domain can also be a combination ofsignaling domains derived from molecules selected from CD3, Fc receptorγ, FcγRIIa, FcRβ. (FcεR1b), CD3γ, CD3δ, CD3ε, CD79a, CD79b, DAP10, andDAP12. A signaling domain derived from a protein or polypeptide refersto the domain of the protein or polypeptide that is responsible foractivating the immune effector cell (e.g., a T cell), or a fragmentthereof that retains its activation function. In general, the signalingdomain induces persistence, trafficking and/or effector functions in thetransduced immune effector cells such as T cells (Sharpe et al., Dis.Model Mech. 8:337-350 (2015); Finney et al., J. Immunol. 161:2791-2797(1998); Krause et al., J. Exp. Med. 188:619-626 (1998)). The signalingdomain of a protein or polypeptide can be the intracellular domain ofthe protein or polypeptide. In some embodiments, the signaling domaincomprises the intracellular domain of CD3ζ, FcRγ, FcγRIIa, FcRβ, CD3γ,CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, DAP10, DAP12, or any combinationthereof.

In some embodiments, the cytoplasmic domain of CARs provided hereincomprises a signaling domain derived from CD3ζ. In some embodiments, thesignaling domain comprises the intracellular domain of CD3ζ. In someembodiments, the cytoplasmic domain comprises a signaling domain derivedfrom FcRγ. In some embodiments, the signaling domain comprises theintracellular domain of FcRγ. In some embodiments, the cytoplasmicdomain comprises a signaling domain derived from FcγRIIa. In someembodiments, the signaling domain comprises the intracellular domain ofFcγRIIa. In some embodiments, the cytoplasmic domain comprises asignaling domain derived from FcRβ. In some embodiments, the signalingdomain comprises the intracellular domain of FcRβ. In some embodiments,the cytoplasmic domain comprises a signaling domain derived from CD3γ.In some embodiments, the signaling domain comprises the intracellulardomain of CD3γ. In some embodiments, the cytoplasmic domain comprises asignaling domain derived from CD3δ. In some embodiments, the signalingdomain comprises the intracellular domain of CD3δ. In some embodiments,the cytoplasmic domain comprises a signaling domain derived from CD3ε.In some embodiments, the signaling domain comprises the intracellulardomain of CD3ε. In some embodiments, the cytoplasmic domain comprises asignaling domain derived from CD5. In some embodiments, the signalingdomain comprises the intracellular domain of CD5. In some embodiments,the cytoplasmic domain comprises a signaling domain derived from CD22.In some embodiments, the signaling domain comprises the intracellulardomain of CD22. In some embodiments, the cytoplasmic domain comprises asignaling domain derived from CD79a. In some embodiments, the signalingdomain comprises the intracellular domain of CD79a. In some embodiments,the cytoplasmic domain comprises a signaling domain derived from CD79b.In some embodiments, the signaling domain comprises the intracellulardomain of CD79b. In some embodiments, the cytoplasmic domain comprises asignaling domain derived from DAP10. In some embodiments, the signalingdomain comprises the intracellular domain of DAP10. In some embodiments,the cytoplasmic domain comprises a signaling domain derived from DAP12.In some embodiments, the signaling domain comprises the intracellulardomain of DAP12. Exemplary signaling domains are described below in moredetail.

In some embodiments, the cytoplasmic domain of CARs provided hereinfurther comprises a co-stimulatory domain. In some embodiments, thecytoplasmic domain of CARs provided herein further comprises twoco-stimulatory domains. Such a co-stimulatory domain can provideincreased activation of an immune effector cell (e.g., T cell). Aco-stimulatory signaling domain can be derived from, for example, CD28,4-1BB (CD137), OX40, ICOS, DAP10, 2B4, CD27, CD30, CD40, CD2, CD7,LIGHT, TIGIT, GITR, TLR, DR3, or CD43. A co-stimulatory domain derivedfrom a protein or polypeptide refers to the domain of the protein orpolypeptide that is responsible for providing increased activation of animmune effector cell (e.g., T cell), or a fragment thereof that retainsits activation function. In some embodiments, the co-stimulatory domainof CARs provided herein comprises the intracellular domain of CD28,4-1BB (CD137), OX40, ICOS, DAP10, 2B4, CD27, CD30, CD40, CD2, CD7,LIGHT, TIGIT, GITR, TLR, DR3, or CD43. In some embodiments, thecytoplasmic domain of CARs provided herein comprises a co-stimulatorydomain derived from CD28. In some embodiments, the co-stimulatory domaincomprises the intracellular domain of CD28. In some embodiments, thecytoplasmic domain comprises a co-stimulatory domain derived from 4-1BB.In some embodiments, the co-stimulatory domain comprises theintracellular domain of 4-1BB. In some embodiments, the cytoplasmicdomain comprises a co-stimulatory domain derived from OX40. In someembodiments, the co-stimulatory domain comprises the intracellulardomain of OX40. In some embodiments, the cytoplasmic domain comprises aco-stimulatory domain derived from ICOS. In some embodiments, theco-stimulatory domain comprises the intracellular domain of ICOS. Insome embodiments, the cytoplasmic domain comprises a co-stimulatorydomain derived from DAP10. In some embodiments, the co-stimulatorydomain comprises the intracellular domain of DAP10. In some embodiments,the cytoplasmic domain comprises a co-stimulatory domain derived from2B4. In some embodiments, the co-stimulatory domain comprises theintracellular domain of 2B4. In some embodiments, the cytoplasmic domaincomprises a co-stimulatory domain derived from CD27. In someembodiments, the co-stimulatory domain comprises the intracellulardomain of CD27. In some embodiments, the cytoplasmic domain comprises aco-stimulatory domain derived from CD30. In some embodiments, theco-stimulatory domain comprises the intracellular domain of CD30. Insome embodiments, the cytoplasmic domain comprises a co-stimulatorydomain derived from CD40. In some embodiments, the co-stimulatory domaincomprises the intracellular domain of CD40. In some embodiments, thecytoplasmic domain comprises a co-stimulatory domain derived from CD2.In some embodiments, the co-stimulatory domain comprises theintracellular domain of CD2. In some embodiments, the cytoplasmic domaincomprises a co-stimulatory domain derived from CD7. In some embodiments,the co-stimulatory domain comprises the intracellular domain of CD7. Insome embodiments, the cytoplasmic domain comprises a co-stimulatorydomain derived from LIGHT. In some embodiments, the co-stimulatorydomain comprises the intracellular domain of LIGHT. In some embodiments,the cytoplasmic domain comprises a co-stimulatory domain derived fromTIGIT. In some embodiments, the co-stimulatory domain comprises theintracellular domain of TIGIT. In some embodiments, the cytoplasmicdomain comprises a co-stimulatory domain derived from GITR. In someembodiments, the co-stimulatory domain comprises the intracellulardomain of GITR. In some embodiments, the cytoplasmic domain comprises aco-stimulatory domain derived from TLR. In some embodiments, theco-stimulatory domain comprises the intracellular domain of TLR. In someembodiments, the cytoplasmic domain comprises a co-stimulatory domainderived from DR3. In some embodiments, the co-stimulatory domaincomprises the intracellular domain of DR3. In some embodiments, thecytoplasmic domain comprises a co-stimulatory domain derived from CD43.In some embodiments, the co-stimulatory domain comprises theintracellular domain of CD43. Exemplary co-stimulatory domains aredescribed below in more detail.

CARs comprising an intracellular domain that comprises a co-stimulatorydomain derived from 4-1BB, ICOS or DAP-10 have been described previously(see U.S. Pat. No. 7,446,190, which is incorporated herein by reference,which also describes representative sequences for 4-1BB, ICOS andDAP-10). In some embodiments, the cytoplasmic domain of a CAR cancomprise two co-stimulatory domains derived from two co-stimulatoryreceptors, such as CD28 and 4-1BB (see Sadelain et al., Cancer Discov.3(4):388-398 (2013)), or CD28 and OX40, or other combinations ofco-stimulatory ligands, as disclosed herein.

The extracellular domain of a CAR can be fused to a leader or a signalpeptide that directs the nascent protein into the endoplasmic reticulumand subsequent translocation to the cell surface. It is understood that,once a polypeptide containing a signal peptide is expressed at the cellsurface, the signal peptide has generally been proteolytically removedduring processing of the polypeptide in the endoplasmic reticulum andtranslocation to the cell surface. Thus, a polypeptide such as a CAR isgenerally expressed at the cell surface as a mature protein lacking thesignal peptide, whereas the precursor form of the polypeptide includesthe signal peptide. A signal peptide or leader can be essential if a CARis to be glycosylated and/or anchored in the cell membrane. The signalsequence or leader is a peptide sequence generally present at theN-terminus of newly synthesized proteins that directs their entry intothe secretory pathway. The signal peptide is covalently joined to theN-terminus of the extracellular antigen-binding domain of a CAR as afusion protein. Any suitable signal peptide, as are well known in theart, can be applied to a CAR to provide cell surface expression in animmune cell (see Gierasch Biochem. 28:923-930 (1989); von Heijne, J.Mol. Biol. 184 (1):99-105 (1985)). Particularly useful signal peptidescan be derived from cell surface proteins naturally expressed in theimmune cell provided herein, including any of the signal peptides of thepolypeptides disclosed herein. Thus, any suitable signal peptide can beutilized to direct a CAR to be expressed at the cell surface of animmune effector cell provided herein.

In some embodiments, a CAR can also comprise a spacer region or sequencethat links the domains of the CAR to each other. For example, a spacercan be included between a signal peptide and an antigen binding domain,between the antigen binding domain and the transmembrane domain, betweenthe transmembrane domain and the intracellular domain, and/or betweendomains within the intracellular domain, for example, between astimulatory domain and a co-stimulatory domain. The spacer region can beflexible enough to allow interactions of various domains with otherpolypeptides, for example, to allow the antigen binding domain to haveflexibility in orientation in order to facilitate antigen recognition.The spacer region can be, for example, the hinge region from an IgG, theCH₂CH₃ (constant) region of an immunoglobulin, and/or portions of CD3(cluster of differentiation 3) or some other sequence suitable as aspacer. In some embodiments, a CAR disclosed herein comprises a hingedomain that connects the BCMA binding domain and the transmembranedomain. In some embodiments, the hinge domain comprises human CD8 hingedomain. In some embodiments, the hinge domain comprises human CD28 hingedomain.

Provided below are some exemplary molecules from which domains of theCARs provided herein can be derived.

CD3ζ. CD3ζ comprises 3 Immune-receptor-Tyrosine-based-Activation-Motifs(ITAMs), and transmits an activation signal to the cell, for example, acell of the lymphoid lineage such as a T cell, after antigen is bound. ACD3ζ polypeptide can have an amino acid sequence corresponding to thesequence having GenBank No. NP_932170 (NP_932170.1, GI:37595565; seebelow), or fragments thereof. In some embodiments, a CD3ζ signalingdomain has an amino acid sequence of amino acids 52 to 164 of the CD3ζpolypeptide sequence provided below, or a fragment thereof that issufficient for signaling activity. See GenBank NP_932170 for referenceto domains within CD3ζ, for example, signal peptide, amino acids 1 to21; extracellular domain, amino acids 22 to 30; transmembrane region,amino acids 31 to 51; intracellular domain, amino acids 52 to 164. Insome embodiments, a CAR can have a transmembrane domain derived fromCD3ζ. The transmembrane domain can comprise the transmembrane region ofCD3ζ (e.g., amino acids 31 to 51 of the sequence below), or a fragmentthereof. In some embodiments, the cytoplasmic domain of a CAR cancomprise a signaling domain derived from CD3ζ. In some embodiments, asignaling domain of CD3ζ can comprise the intracellular domain of CD3ζ(e.g., amino acids 52 to 164 of the sequence below), or a fragmentthereof. It is understood that sequences of CD3ζ that are shorter orlonger than a specific delineated domain can be included in a CAR, ifdesired.

(SEQ ID NO: 21)  1 MKWKALFTAA ILQAQLPITE AQSFGLLDPKLCYLLDGILF IYGVILTALF LRVKFSRSAD 61 APAYQQGQNQ LYNELNLGRR EEYDVLDKRRGRDPEMGGKP QRRKNPQEGL YNELQKDKMA 121 EAYSEIGMKG ERRRGKGHDG LYQGLSTATKDTYDALHMQA LPPR

FcRγ Activating types of IgG receptor FcγRs form multimeric complexesincluding the Fc receptor common γ chain (FcRγ) that contains anintracellular tyrosine-based activating motif (ITAM), whose activationtriggers oxidative bursts, cytokine release, phagocytosis,antibody-dependent cell-mediated cytotoxicity, and degranulation. AnFcRγ polypeptide can have an amino acid sequence corresponding to thesequence having NCBI Reference Sequence: NP_004097.1 (GI: 4758344), orfragments thereof. See GenBank NP_004097 for reference to domains withinFcRγ, for example, signal peptide, amino acids 1 to 18; extracellulardomain, amino acids 19 to 23; transmembrane region, amino acids 24 to44; intracellular domain, amino acids 45 to 86. In some embodiments, aCAR can comprise a transmembrane domain derived from FcRγ. In someembodiments, the transmembrane domain of the CAR comprises thetransmembrane region of FcRγ, or a fragment thereof. In someembodiments, the cytoplasmic domain of a CAR can comprise a signalingdomain derived from FcRγ. In some embodiments, the signaling domaincomprises the intracellular domain of FcRγ, or a fragment thereof. It isunderstood that sequences of FcRγ that are shorter or longer than aspecific delineated domain can be included in a CAR, if desired.

FcγRIIa is a cell surface receptor found on phagocytic cells such asmacrophages and neutrophils, and is involved in the process ofphagocytosis and clearing of immune complexes. By binding to IgG itinitiates cellular responses against pathogens and soluble antigens.FcγRIIa also promotes phagocytosis of opsonized antigens. An FcγRIIapolypeptide can have an amino acid sequence corresponding to thesequence having NCBI Reference Sequence: NP_001129691.1, or fragmentsthereof. See NCBI Reference Sequence NP_001129691.1 for reference todomains within FcγRIIa, for example, signal peptide, amino acids 1 to33; extracellular domain, amino acids 34 to 217; transmembrane region,amino acids 218 to 240; intracellular domain, amino acids 241 to 317. Insome embodiments, a CAR can comprise a transmembrane domain derived fromFcγRIIa. In some embodiments, the transmembrane domain of the CARcomprises the transmembrane region of FcγRIIa, or a fragment thereof. Insome embodiments, the cytoplasmic domain of a CAR can comprise asignaling domain derived from FcγRIIa. In some embodiments, thesignaling domain comprises the intracellular domain of FcγRIIa, or afragment thereof. It is understood that sequences of FcγRIIa that areshorter or longer than a specific delineated domain can be included in aCAR, if desired.

FcRβ (FcεR1b) is a high affinity receptor that binds to the Fc region ofimmunoglobulins epsilon. Aggregation of FcRβ by multivalent antigens isrequired for the full mast cell response, including the release ofpreformed mediators (such as histamine) by degranulation and de novoproduction of lipid mediators and cytokines. FcRβ also mediates thesecretion of important lymphokines. Binding of allergen toreceptor-bound IgE leads to cell activation and the release of mediatorsresponsible for the manifestations of allergy. An FcRβ polypeptide canhave an amino acid sequence corresponding to the sequence having NCBIReference Sequence: NP_000130.1, or fragments thereof. See NCBIReference Sequence: NP_000130.1 for reference to domains within FcRβ,for example, intracellular domain, amino acids 1 to 59, 118 to 130, and201 to 244; transmembrane region, amino acids 60 to 79, 98 to 117, 131to 150, and 181 to 200; extracellular domain, amino acids 80 to 97, and151 to 180. In some embodiments, the cytoplasmic domain of a CAR cancomprise a signaling domain derived from FcRβ. In some embodiments, thesignaling domain comprises an intracellular domain of FcRβ, or afragment thereof. It is understood that sequences of FcRβ that areshorter or longer than a specific delineated domain can be included in aCAR, if desired.

CD3γ (T-cell surface glycoprotein CD3 gamma chain), is part of theTCR-CD3 complex present on T-lymphocyte cell surface that plays anessential role in adaptive immune response. CD3γ contains immunoreceptortyrosine-based activation motifs (ITAMs) in its cytoplasmic domain. Inaddition to this role of signal transduction in T-cell activation, CD3γplays an essential role in the dynamic regulation of TCR expression atthe cell surface. A CD3γ polypeptide can have an amino acid sequencecorresponding to the sequence having NCBI Reference Sequence:NP_004097.1 (GI: 4758344), or fragments thereof. See GenBank NP_004097for reference to domains within CD3γ, for example, signal peptide, aminoacids 1 to 22; extracellular domain, amino acids 23 to 116;transmembrane region, amino acids 117 to 137; intracellular domain,amino acids 138 to 182. In some embodiments, a CAR can comprise atransmembrane domain derived from CD3γ. In some embodiments, thetransmembrane domain of the CAR comprises the transmembrane region ofCD3γ, or a fragment thereof. In some embodiments, the cytoplasmic domainof a CAR can comprise a signaling domain derived from CD3γ. In someembodiments, the signaling domain comprises the intracellular domain ofCD3γ, or a fragment thereof. It is understood that sequences of CD3γthat are shorter or longer than a specific delineated domain can beincluded in a CAR, if desired.

CD3δ (T-cell surface glycoprotein CD3 delta chain), is part of theTCR-CD3 complex present on T-lymphocyte cell surface that plays anessential role in adaptive immune response. CD3δ contains immunoreceptortyrosine-based activation motifs (ITAMs) in its cytoplasmic domain. Inaddition of this role of signal transduction in T-cell activation, CD3δplays an essential role in thymocyte differentiation and participates incorrect intracellular TCR-CD3 complex assembly and surface expression.CD3δ interacts with CD4 and CD8 and thus serves to establish afunctional link between the TCR and coreceptors CD4 and CD8, which isneeded for activation and positive selection of CD4 or CD8 T-cells. ACD3δ polypeptide can have an amino acid sequence corresponding to thesequence having NCBI Reference Sequence: NP_000723.1, or fragmentsthereof. See NCBI Reference Sequence: NP_000723.1 for reference todomains within CD3δ, for example, signal peptide, amino acids 1 to 21;extracellular domain, amino acids 22 to 105; transmembrane region, aminoacids 106 to 126; intracellular domain, amino acids 127 to 171. In someembodiments, a CAR can comprise a transmembrane domain derived fromCD3δ. In some embodiments, the transmembrane domain of the CAR comprisesthe transmembrane region of CD3δ, or a fragment thereof. In someembodiments, the cytoplasmic domain of a CAR can comprise a signalingdomain derived from CD3δ. In some embodiments, the signaling domaincomprises the intracellular domain of CD3δ, or a fragment thereof. It isunderstood that sequences of CD3δ that are shorter or longer than aspecific delineated domain can be included in a CAR, if desired.

CD3ε (T-cell surface glycoprotein CD3 epsilon chain), is part of theTCR-CD3 complex present on T-lymphocyte cell surface that plays anessential role in adaptive immune response. CD3ε contains immunoreceptortyrosine-based activation motifs (ITAMs) in its cytoplasmic domain. Inaddition of this role of signal transduction in T-cell activation, CD3εplays an essential role in correct T-cell development. CDR3ε initiatesthe TCR-CD3 complex assembly by forming the two heterodimers CD3δ/CD3γand CD3γ/CD3γ. A CD3ε polypeptide can have an amino acid sequencecorresponding to the sequence having NCBI Reference Sequence:NP_000724.1, or fragments thereof. See NCBI Reference Sequence:NP_000724.1 for reference to domains within CD3ε, for example, signalpeptide, amino acids 1 to 22; extracellular domain, amino acids 23 to126; transmembrane region, amino acids 127 to 152; intracellular domain,amino acids 153 to 207. In some embodiments, a CAR can comprise atransmembrane domain derived from CD3ε. In some embodiments, thetransmembrane domain of the CAR comprises the transmembrane region ofCD3ε, or a fragment thereof. In some embodiments, the cytoplasmic domainof a CAR can comprise a signaling domain derived from CD3ε. In someembodiments, the signaling domain comprises the intracellular domain ofCD3ε, or a fragment thereof. It is understood that sequences of CD3εthat are shorter or longer than a specific delineated domain can beincluded in a CAR, if desired.

CD79a (B-cell antigen receptor complex-associated protein alpha chain)is required in cooperation with CD79b for initiation of the signaltransduction cascade activated by binding of antigen to the B-cellantigen receptor complex (BCR) which leads to internalization of thecomplex, trafficking to late endosomes and antigen presentation. CD79astimulates SYK autophosphorylation and activation. CD79a also binds toBLNK, bringing BLNK into proximity with SYK and allowing SYK tophosphorylate BLNK, and interacts with and increases activity of someSrc-family tyrosine kinases. A CD79a polypeptide can have an amino acidsequence corresponding to the sequence having NCBI Reference Sequence:NP_001774.1, or fragments thereof. See NCBI Reference Sequence:NP_001774.1 for reference to domains within CD79a, for example, signalpeptide, amino acids 1 to 32; extracellular domain, amino acids 33 to143; transmembrane region, amino acids 144 to 165; intracellular domain,amino acids 166 to 226. In some embodiments, a CAR can comprise atransmembrane domain derived from CD79a. In some embodiments, thetransmembrane domain of the CAR comprises the transmembrane region ofCD79a, or a fragment thereof. In some embodiments, the cytoplasmicdomain of a CAR can comprise a signaling domain derived from CD79a. Insome embodiments, the signaling domain comprises the intracellulardomain of CD79a, or a fragment thereof. It is understood that sequencesof CD79a that are shorter or longer than a specific delineated domaincan be included in a CAR, if desired.

CD79b (B-cell antigen receptor complex-associated protein beta chain) isrequired in cooperation with CD79a for initiation of the signaltransduction cascade activated by the B-cell antigen receptor complex(BCR) which leads to internalization of the complex, trafficking to lateendosomes and antigen presentation. CD79b enhances phosphorylation ofCD79a. A CD79b polypeptide can have an amino acid sequence correspondingto the sequence having NCBI Reference Sequence: NP_000617.1, orfragments thereof. See NCBI Reference Sequence: NP_000617.1 forreference to domains within CD79b, for example, signal peptide, aminoacids 1 to 28; extracellular domain, amino acids 29 to 159;transmembrane region, amino acids 160 to 180; intracellular domain,amino acids 181 to 229. In some embodiments, a CAR can comprise atransmembrane domain derived from CD79b. In some embodiments, thetransmembrane domain of the CAR comprises the transmembrane region ofCD79b, or a fragment thereof. In some embodiments, the cytoplasmicdomain of a CAR can comprise a signaling domain derived from CD79b. Insome embodiments, the signaling domain comprises the intracellulardomain of CD79b, or a fragment thereof. It is understood that sequencesof CD79b that are shorter or longer than a specific delineated domaincan be included in a CAR, if desired.

DAP10. DAP10, also referred to as hematopoietic cell signal transducer,is a signaling subunit that associates with a large family of receptorsin hematopoietic cells. A DAP10 polypeptide can have an amino acidsequence corresponding to the sequence having GenBank No. NP_055081.1(GI:15826850), or fragments thereof. See GenBank NP_055081 for referenceto domains within DAP10, for example, signal peptide, amino acids 1 to18; extracellular domain, amino acids 19 to 48; transmembrane region,amino acids 49 to 69; intracellular domain, amino acids 70 to 93. Insome embodiments, the cytoplasmic domain of a CAR can comprise asignaling domain derived from DAP10. In some embodiments, the signalingdomain comprises the intracellular domain of DAP10, or a fragmentthereof. In some embodiments, the cytoplasmic domain comprises aco-stimulatory domain derived from DAP10. In some embodiments, theco-stimulatory domain comprises the intracellular domain of DAP10, or afragment thereof. It is understood that sequences of DAP10 that areshorter or longer than a specific delineated domain can be included in aCAR, if desired.

DAP12. DAP12 is found in cells of the myeloid lineage, such asmacrophages and granulocytes, where it associates, for instance, withthe triggering receptor expressed on myeloid cell members (TREM) andMDL1 (myeloid DAP12-associating lectin 1/CLEC5A), both involved ininflammatory responses against pathogens like viruses and bacteria. Inthe lymphoid lineage, DAP12 is expressed in NK cells and associates withactivating receptors such as the C-type lectin receptor NKG2C, thenatural cytotoxicity receptor NKp44, and the short-tailed KIR3DS1 andKIR2DS1/2/5, respectively. In particular, NGK2C is the dominantactivating NK cell receptor for controlling CMV infection in both humansand mice. It was found that a DAP12-containing CAR generated sufficientactivating signals in NK cells upon cross-linking with its Ag. Töpfer etal., J Immunol 194:3201-12 (2015). A DAP12 polypeptide can have an aminoacid sequence corresponding to the sequence having GenBank No.AAD09437.1 (GI: 2905996), or fragments thereof. See GenBank No.AAD09437.1 for reference to domains within DAP12, for example, signalpeptide, amino acids 1 to 21; extracellular domain, amino acids 22 to40; transmembrane region, amino acids 41 to 61; intracellular domain,amino acids 62 to 113. In some embodiments, the cytoplasmic domain of aCAR can comprise a signaling domain derived from DAP12. In someembodiments, the signaling domain comprises the intracellular domain ofDAP12, or a fragment thereof. In some embodiments, the cytoplasmicdomain comprises a co-stimulatory domain derived from DAP12. In someembodiments, the co-stimulatory domain comprises the intracellulardomain of DAP12, or a fragment thereof. It is understood that sequencesof DAP12 that are shorter or longer than a specific delineated domaincan be included in a CAR, if desired.

CD28. Cluster of Differentiation 28 (CD28) is a protein expressed on Tcells that provides co-stimulatory signals for T cell activation andsurvival. CD28 is the receptor for CD80 (B7.1) and CD86 (B7.2) proteins.A CD28 polypeptide can have an amino acid sequence corresponding to thesequence having GenBank No. P10747 (P10747.1, GI:115973) or NP_006130(NP_006130.1, GI:5453611), as provided below, or fragments thereof. SeeGenBank NP_006130 for reference to domains within CD28, for example,signal peptide, amino acids 1 to 18; extracellular domain, amino acids19 to 152; transmembrane domain, amino acids 153 to 179; intracellulardomain, amino acids 180 to 220. In some embodiments, a CAR can comprisea hinge domain derived from CD28 (e.g., amino acids 114 to 152 of thesequence below, or a fragment thereof). In some embodiments, a CAR cancomprise a transmembrane domain derived from CD28. In some embodiments,the transmembrane domain of the CAR comprises the transmembrane regionof CD28 (e.g., amino acids 153 to 179 of the sequence below), or afragment thereof. In some embodiments, the cytoplasmic domain of a CARcan comprise a co-stimulatory domain derived from CD28. In someembodiments, the co-stimulatory domain comprises the intracellulardomain of CD28 (e.g., amino acids 180 to 220 of the sequence below), ora fragment thereof. In some embodiments, a CAR can comprise two domainsderived from CD28, a co-stimulatory signaling domain and a transmembranedomain. In some embodiments, a CAR has an amino acid sequence comprisingthe transmembrane domain and the intracellular domain of CD28 andcomprises amino acids 153 to 220 of CD28. In some embodiments, a CAR cancomprise three domains derived from CD28, a transmembrane domain, ahinge domain and a co-stimulatory signaling domain. In anotherembodiment, a CAR comprises amino acids 114 to 220 of CD28. It isunderstood that sequences of CD28 that are shorter or longer than aspecific delineated domain can be included in a CAR, if desired.

(SEQ ID NO: 22) 1 MLRLLLALNL FPSIQVTGNK ILVKQSPMLVAYDNAVNLSC KYSYNLFSRE FRASLHKGLD 61 SAVEVCVVYGNYSQQLQVYS KTGFNCDGKLGNESVTFYLQ NLYVNQTDIY FCKIEVMYPP 121 PYLDNEKSNG TIIHVKGKHL CPSPLFPGPSKPFWVLVVVG GVLACYSLLV TVAFIIFWVR 181 SKRSRLLHSD YMNMTPRRPG PTRKHYQPYAPPRDFAAYRS

4-1BB. 4-1BB, also referred to as tumor necrosis factor receptorsuperfamily member 9, can act as a tumor necrosis factor (TNF) ligandand have stimulatory activity. A 4-1BB polypeptide can have an aminoacid sequence corresponding to the sequence having GenBank No. P41273(P41273.1, GI:728739) or NP_001552 (NP_001552.2, GI:5730095) orfragments thereof. See GenBank NP_001552 for reference to domains within4-1BB, for example, signal peptide, amino acids 1 to 17; extracellulardomain, amino acids 18 to 186; transmembrane domain, amino acids 187 to213; intracellular domain, amino acids 214 to 255. In some embodiments,a CAR can comprise a transmembrane domain derived from 4-1BB. In someembodiments, the transmembrane domain of the CAR comprises thetransmembrane region of 4-1BB (e.g., amino acids 187 to 213 of thesequence below), or a fragment thereof. In some embodiments, thecytoplasmic domain of a CAR can comprise a co-stimulatory domain derivedfrom 4-1BB. In some embodiments, the co-stimulatory domain comprises theintracellular domain of 4-1BB (e.g., amino acids 214 to 255 of thesequence below), or a fragment thereof. In some embodiments, a CAR cancomprise two domains derived from 4-1BB, a co-stimulatory signalingdomain and a transmembrane domain. In some embodiments, a CAR has anamino acid sequence comprising the transmembrane domain and theintracellular domain of 4-1BB and comprises amino acids 187 to 255 of4-1BB. It is understood that sequences of 4-1BB that are shorter orlonger than a specific delineated domain can be included in a CAR, ifdesired.

(SEQ ID NO: 23) 1 MGNSCYNIVA TLLLVLNFER TRSLQDPCSNCPAGTFCDNN RNQICSPCPP NSFSSAGGQR 61 TCDICRQCKG VFRTRKECSS TSNAECDCTPGFHCLGAGCS MCEQDCKQGQ ELTKKGCKDC 121 CFGTFNDQKR GICRPWTNCS LDGKSVLVNGTKERDVVCGP SPADLSPGAS SVTPPAPARE 181 PGHSPQIISF FLALTSTALL FLLFFLTLRFSVVKRGRKKL LYIFKQPFMR PVQTTQEEDG 241 CSCRFPEEEE GGCEL

OX40. OX40, also referred to as tumor necrosis factor receptorsuperfamily member 4 precursor or CD134, is a member of theTNFR-superfamily of receptors. An OX40 polypeptide can have an aminoacid sequence corresponding to the sequence having GenBank No. P43489(P43489.1, GI:1171933) or NP_003318 (NP_003318.1, GI:4507579), orfragments thereof. See GenBank NP_003318 for reference to domains withinOX40, for example, signal peptide, amino acids 1 to 28; extracellulardomain, amino acids 29 to 214; transmembrane domain, amino acids 215 to235; intracellular domain, amino acids 236 to 277. It is understood thatsequences of OX40 that are shorter or longer than a specific delineateddomain can be included in a CAR, if desired. In some embodiments, a CARcan comprise a transmembrane domain derived from OX40. In someembodiments, the transmembrane domain of the CAR comprises thetransmembrane region of OX40, or a fragment thereof. In someembodiments, the cytoplasmic domain of a CAR can comprise aco-stimulatory domain derived from OX40. In some embodiments, theco-stimulatory domain comprises the intracellular domain of OX40, or afragment thereof. In some embodiments, a CAR can comprise two domainsderived from OX40, a co-stimulatory signaling domain and a transmembranedomain. In some embodiments, a CAR has an amino acid sequence comprisingthe transmembrane domain and the intracellular domain of OX40 andcomprises amino acids 215 to 277 of OX40.

ICOS. Inducible T-cell co-stimulator precursor (ICOS), also referred toas CD278, is a CD28-superfamily co-stimulatory receptor that isexpressed on activated T cells. An ICOS polypeptide can have an aminoacid sequence corresponding to the sequence having GenBank No. NP_036224(NP_036224.1, GI:15029518), or fragments thereof. See GenBank NP_036224for reference to domains within ICOS, for example, signal peptide, aminoacids 1 to 20; extracellular domain, amino acids 21 to 140;transmembrane domain, amino acids 141 to 161; intracellular domain,amino acids 162 to 199. In some embodiments, a CAR can comprise atransmembrane domain derived from ICOS. In some embodiments, thetransmembrane domain of the CAR comprises the transmembrane region ofICOS, or a fragment thereof. In some embodiments, the cytoplasmic domainof a CAR can comprise a co-stimulatory domain derived from ICOS. In someembodiments, the co-stimulatory domain comprises the intracellulardomain of ICOS, or a fragment thereof. In some embodiments, a CAR cancomprise two domains derived from ICOS, a co-stimulatory signalingdomain and a transmembrane domain. In some embodiments, a CAR has anamino acid sequence comprising the transmembrane domain and theintracellular domain of ICOS and comprises amino acids 141 to 199 ofICOS. It is understood that sequences of ICOS that are shorter or longerthan a specific delineated domain can be included in a CAR, if desired.

2B4 2B4 (CD244) is a co-stimulatory receptor expressed on both NK cellsand CD8+ T cells. It targets a non-MHC like molecule (CD48) expressed onhematopoietic cells, including B and T cells, as well as on activatedmonocytes and granulocytes. Activation of 2B4 by binding of its ligandon target cells leads to NK (or T cell) activation, and target killing.A 2B4 polypeptide can have an amino acid sequence corresponding to thesequence having Accession No: Q9BZW8.2 (NP_001160135.1; GI: 47605541),or fragments thereof. See GenBank NP_001160135.1 for reference todomains within 2B4, for example, signal peptide, amino acids 1 to 21;extracellular domain, amino acids 22 to 229; transmembrane domain, aminoacids 230 to 250; intracellular domain, amino acids 251 to 370. In someembodiments, a CAR can comprise a transmembrane domain derived from 2B4.In some embodiments, the transmembrane domain of the CAR comprises thetransmembrane region of 2B4, or a fragment thereof. In some embodiments,the cytoplasmic domain of a CAR can comprise a co-stimulatory domainderived from 2B4. In some embodiments, the co-stimulatory domaincomprises the intracellular domain of 2B4, or a fragment thereof. Insome embodiments, a CAR can comprise two domains derived from 2B4, aco-stimulatory signaling domain and a transmembrane domain. In someembodiments, a CAR has an amino acid sequence comprising thetransmembrane domain and the intracellular domain of 2B4 and comprisesamino acids 230 to 370 of 2B4. It is understood that sequences of 2B4that are shorter or longer than a specific delineated domain can beincluded in a CAR, if desired.

CD27: CD27 (TNFRSF7) is a transmembrane receptor expressed on subsets ofhuman CD8+ and CD4+ T-cells, NKT cells, NK cell subsets andhematopoietic progenitors and induced in FOXP3+ CD4 T-cells and B cellsubsets. Previously studies have found that CD27 can either activelyprovide costimulatory signals that improve human T-cell survival andanti-tumor activity in vivo. (See Song and Powell; Oncoimmunology 1, no.4 (2012): 547-549). A CD27 polypeptide can have an amino acid sequencecorresponding to the sequence having UniProtKB/Swiss-Prot No.: P26842.2(GenBank NP_001233.1; GI: 269849546), or fragments thereof. See GenBankNP_001233 for reference to domains within CD27, for example, signalpeptide, amino acids 1 to 19; extracellular domain, amino acids 20 to191; transmembrane domain, amino acids 192 to 212; intracellular domain,amino acids 213 to 260. In some embodiments, a CAR can comprise atransmembrane domain derived from CD27. In some embodiments, thetransmembrane domain of the CAR comprises the transmembrane region ofCD27, or a fragment thereof. In some embodiments, the cytoplasmic domainof a CAR can comprise a co-stimulatory domain derived from CD27. In someembodiments, the co-stimulatory domain comprises the intracellulardomain of CD27, or a fragment thereof. In some embodiments, a CAR cancomprise two domains derived from CD27, a co-stimulatory signalingdomain and a transmembrane domain. In some embodiments, a CAR has anamino acid sequence comprising the transmembrane domain and theintracellular domain of CD27 and comprises amino acids 192 to 260 ofCD27. It is understood that sequences of CD27 that are shorter or longerthan a specific delineated domain can be included in a CAR, if desired.

CD30: CD30 and its ligand (CD30L) are members of the tumor necrosisfactor receptor (TNFR) and tumor necrosis factor (TNF) superfamilies,respectively. CD30, in many respects, behaves similarly to Ox40 andenhances proliferation and cytokine production induced by TCRstimulation. (Goronzy and Weyand, Arthritis research & therapy 10, no.S1 (2008): S3.) A CD30 polypeptide can have an amino acid sequencecorresponding to the sequence having GenBank No.: AAA51947.1 (GenBankNP_001234.3; GI: 180096), or fragments thereof. See GenBank NP_001234.3for reference to domains within CD30, for example, signal peptide, aminoacids 1 to 18; extracellular domain, amino acids 19 to 385;transmembrane domain, amino acids 386 to 406; intracellular domain,amino acids 407 to 595. In some embodiments, a CAR can comprise atransmembrane domain derived from CD30. In some embodiments, thetransmembrane domain of the CAR comprises the transmembrane region ofCD30, or a fragment thereof. In some embodiments, the cytoplasmic domainof a CAR can comprise a co-stimulatory domain derived from CD30. In someembodiments, the co-stimulatory domain comprises the intracellulardomain of CD30, or a fragment thereof. In some embodiments, a CAR cancomprise two domains derived from CD30, a co-stimulatory signalingdomain and a transmembrane domain. In some embodiments, a CAR has anamino acid sequence comprising the transmembrane domain and theintracellular domain of CD30 and comprises amino acids 386 to 595 ofCD30. It is understood that sequences of CD30 that are shorter or longerthan a specific delineated domain can be included in a CAR, if desired.

CD40: CD40 is a 48 kD transmembrane glycoprotein surface receptor thatis a member of the Tumor Necrosis Factor Receptor superfamily (TNFRSF).Exemplary amino acid sequences of human CD40 are described (see, e.g.,Accession: ALQ33424.1, GenBank NP_001241.1,GI: 957949089), CD40 wasinitially characterized as a co-stimulatory receptor expressed on APCsthat played a central role in B and T cell activation. The ligand forCD40, CD154 (also known as TRAP, T-BAM, CD40 Ligand or CD40L) is a typeII integral membrane protein. See GenBank NP_001241.1 for reference todomains within CD40, for example, signal peptide, amino acids 1 to 20;extracellular domain, amino acids 21 to 193; transmembrane domain, aminoacids 194 to 215; intracellular domain, amino acids 216 to 277. In someembodiments, a CAR can comprise a transmembrane domain derived fromCD40. In some embodiments, the transmembrane domain of the CAR comprisesthe transmembrane region of CD40, or a fragment thereof. In someembodiments, the cytoplasmic domain of a CAR can comprise aco-stimulatory domain derived from CD40. In some embodiments, theco-stimulatory domain comprises the intracellular domain of CD40, or afragment thereof. In some embodiments, a CAR can comprise two domainsderived from CD40, a co-stimulatory signaling domain and a transmembranedomain. In some embodiments, a CAR has an amino acid sequence comprisingthe transmembrane domain and the intracellular domain of CD40 andcomprises amino acids 194 to 277 of CD40. It is understood thatsequences of CD40 that are shorter or longer than a specific delineateddomain can be included in a CAR, if desired.

CD2 The engagement of the CD2 molecule by its ligand CD58 co-stimulatesproliferation, cytokine production, and effector function in this Tcells, especially the CD28-deficient T cells subset. CD58 is broadlyexpressed on APCs including dendritic cells. Engagement of CD2 amplifiesTCR signals in CD28⁻CD8⁺ T cells, demonstrating that the CD2-CD58interaction has a genuine costimulatory effect. CD2 signals couldpromote the control of viral infection by CD28⁻CD8⁺ T cells, but theycould also contribute to the continuous expansion of CD28⁻CD8⁺ T cellsduring chronic stimulation by persistent Ag. (Judith Leitner J et al.,Immunol, 2015, 195 (2) 477-487). A CD2 polypeptide can have an aminoacid sequence corresponding to the sequence having Accession:NP_001758.2 GI: 156071472, or fragments thereof. See GenBank NP_001758.2for reference to domains within CD2, for example, signal peptide, aminoacids 1 to 24; extracellular domain, amino acids 25 to 209;transmembrane domain, amino acids 210 to 235; intracellular domain,amino acids 236 to 351. In some embodiments, a CAR can comprise atransmembrane domain derived from CD2. In some embodiments, thetransmembrane domain of the CAR comprises the transmembrane region ofCD2, or a fragment thereof. In some embodiments, the cytoplasmic domainof a CAR can comprise a co-stimulatory domain derived from CD2. In someembodiments, the co-stimulatory domain comprises the intracellulardomain of CD2, or a fragment thereof. In some embodiments, a CAR cancomprise two domains derived from CD2, a co-stimulatory domain and atransmembrane domain. In some embodiments, a CAR has an amino acidsequence comprising the transmembrane domain and the intracellulardomain of CD2 and comprises amino acids 210 to 351 of CD2. It isunderstood that sequences of CD2 that are shorter or longer than aspecific delineated domain can be included in a CAR, if desired.

LIGHT TNF superfamily member 14 (also known as LTg, CD258, HVEML, LIGHT)is a co-stimulatory receptor involved in cellular immune responses.LIGHT can function as a costimulatory factor for the activation oflymphoid cells and as a deterrent to infection by herpesvirus. LIGHT hasbeen shown to stimulate the proliferation of T cells, and triggerapoptosis of various tumor cells. LIGHT is found in T cells and stromalcells. LIGHT is expressed on immature dendritic cells (DCs) generatedfrom human PBMCs. Engagement of LIGHT co-stimulates human T cellproliferation, amplifies the NF-κB signaling pathway, and preferentiallyinduces the production of IFN-γ, but not IL-4, in the presence of anantigenic signal. (Tamada K et al., J Immunol, 2000, 164 (8) 4105-4110).A LIGHT polypeptide can have an amino acid sequence corresponding to thesequence provided as Accession: NP_001363816.1 GI: 1777376047, orfragments thereof. See GenBank NP_001363816.1 for reference to domainswithin LIGHT, for example, intracellular domain, amino acids 1 to 37;transmembrane domain, amino acids 38 to 58; extracellular domain, aminoacids 59 to 240. In some embodiments, the cytoplasmic domain of a CARcan comprise a co-stimulatory domain derived from LIGHT. In someembodiments, the co-stimulatory domain comprises the intracellulardomain of LIGHT, or a fragment thereof. It is understood that sequencesof LIGHT that are shorter or longer than a specific delineated domaincan be included in a CAR, if desired.

GITR TNF receptor superfamily member 18 (also known as TNFRSF18, AITR,GITR; CD357; GITR-D; ENERGEN) has been shown to have increasedexpression upon T-cell activation. Stimulation of T cells through GITRhas been shown to enhance immunity to tumors and viral pathogens, and toexacerbate autoimmune disease. The effects of stimulation through GITRare generally thought to be caused by attenuation of the effectoractivity of immunosuppressive CD4+CD25+ regulatory T (TReg) cells.(Shevach, E. and Stephens, G. Nat Rev Immunol 6, 613-618 (2006)). A GITRpolypeptide can have an amino acid sequence corresponding to thesequence provided as Accession: AAI52382.1, GenBank NP_004186.1, GI:158931986, or fragments thereof. See GenBank NP_004186.1 for referenceto domains within GITR, for example, signal peptide, amino acids 1 to25; extracellular domain, amino acids 26 to 162; transmembrane domain,amino acids 163 to 183; intracellular domain, amino acids 184 to 241. Insome embodiments, a CAR can comprise a transmembrane domain derived fromGITR. In some embodiments, the transmembrane domain of the CAR comprisesthe transmembrane region of GITR, or a fragment thereof. In someembodiments, the cytoplasmic domain of a CAR can comprise aco-stimulatory domain derived from GITR. In some embodiments, theco-stimulatory domain comprises the intracellular domain of GITR, or afragment thereof. In some embodiments, a CAR can comprise two domainsderived from GITR, a co-stimulatory signaling domain and a transmembranedomain. In some embodiments, a CAR has an amino acid sequence comprisingthe transmembrane domain and the intracellular domain of GITR andcomprises amino acids 163 to 241 of GITR. It is understood thatsequences of GITR that are shorter or longer than a specific delineateddomain can be included in a CAR, if desired.

DR3 TNF receptor superfamily member 25 (also known as DR3, TR3, DDR3,LARD, APO-3, TRAMP, WSL-1, GEF720, WSL-LR, PLEKHG5, or TNFRSF12) isexpressed preferentially in the tissues enriched in lymphocytes, and itplays a role in regulating lymphocyte homeostasis. This receptor hasbeen shown to stimulate NF-kappa B activity and regulate cell apoptosis.The signal transduction of this receptor is mediated by various deathdomain containing adaptor proteins. Multiple alternatively splicedtranscript variants of this gene encoding distinct isoforms have beenreported, most of which are potentially secreted molecules. Thealternative splicing of this gene in B and T cells encounters aprogrammed change upon T-cell activation, which predominantly producesfull-length, membrane bound isoforms, and is involved in controllinglymphocyte proliferation induced by T-cell activation. A DR3 polypeptidecan have an amino acid sequence corresponding to the sequence providedas Accession: Accession: Accession: AAI17190.1, GenBank NP_003781.1 GI:109658976, or fragments thereof. See GenBank NP_003781.1 for referenceto domains within DR3, for example, signal peptide, amino acids 1 to 24;extracellular domain, amino acids 25 to 199; transmembrane domain, aminoacids 200 to 220; intracellular domain, amino acids 221 to 417. In someembodiments, a CAR can comprise a transmembrane domain derived from DR3.In some embodiments, the transmembrane domain of the CAR comprises thetransmembrane region of DR3, or a fragment thereof. In some embodiments,the cytoplasmic domain of a CAR can comprise a co-stimulatory domainderived from DR3. In some embodiments, the co-stimulatory domaincomprises the intracellular domain of DR3, or a fragment thereof. Insome embodiments, a CAR can comprise two domains derived from DR3, aco-stimulatory signaling domain and a transmembrane domain. In someembodiments, a CAR has an amino acid sequence comprising thetransmembrane domain and the intracellular domain of DR3 and comprisesamino acids 200 to 417 of DR3. It is understood that sequences of DR3that are shorter or longer than a specific delineated domain can beincluded in a CAR, if desired.

CD43 CD43 (also known as SPN sialophorin, LSN, GALGP, GPL115) is ahighly sialylated glycoprotein that functions in antigen-specificactivation of T cells, and is found on the surface of thymocytes, Tlymphocytes, monocytes, granulocytes, and some B lymphocytes. Itcontains a mucin-like extracellular domain, a transmembrane region and acarboxy-terminal intracellular region. In stimulated immune effectorcells, proteolytic cleavage of the extracellular domain occurs in somecell types, releasing a soluble extracellular fragment. A CD43polypeptide can have an amino acid sequence corresponding to thesequence provided as GenBank NP_003114.1, Accession: EAW80016.1 GI:119600422, or fragments thereof. See GenBank NP_003114.1 for referenceto domains within CD43, for example, signal peptide, amino acids 1 to19; extracellular domain, amino acids 20 to 253; transmembrane domain,amino acids 254 to 276; intracellular domain, amino acids 277 to 400. Insome embodiments, a CAR can comprise a transmembrane domain derived fromCD43. In some embodiments, the transmembrane domain of the CAR comprisesthe transmembrane region of CD43, or a fragment thereof. In someembodiments, the cytoplasmic domain of a CAR can comprise aco-stimulatory domain derived from CD43. In some embodiments, theco-stimulatory domain comprises the intracellular domain of CD43, or afragment thereof. In some embodiments, a CAR can comprise two domainsderived from CD43, a co-stimulatory signaling domain and a transmembranedomain. In some embodiments, a CAR has an amino acid sequence comprisingthe transmembrane domain and the intracellular domain of CD43 andcomprises amino acids 254 to 400 of CD43. It is understood thatsequences of CD43 that are shorter or longer than a specific delineateddomain can be included in a CAR, if desired.

CD4. Cluster of differentiation 4 (CD4), also referred to as T-cellsurface glycoprotein CD4, is a glycoprotein found on the surface ofimmune cells such as T helper cells, monocytes, macrophages, anddendritic cells. In some embodiments, a CAR can comprise a transmembranedomain derived from CD4. CD4 exists in various isoforms. It isunderstood that any isoform can be selected to achieve a desiredfunction. Exemplary isoforms include isoform 1 (NP_000607.1,GI:10835167), isoform 2 (NP_001181943.1, GI:303522479), isoform 3(NP_001181944.1, GI:303522485; or NP_001181945.1, GI:303522491; orNP_001181946.1, GI:303522569). See GenBank NP_000607.1 for reference todomains within CD4, for example, signal peptide, amino acids 1 to 25;extracellular domain, amino acids 26 to 396; transmembrane domain aminoacids, 397 to 418; intracellular domain, amino acids 419 to 458. In someembodiments, a CAR can comprise a transmembrane domain derived from CD4.In some embodiments, the transmembrane domain of the CAR comprises thetransmembrane region of CD4, or a fragment thereof. It is understoodthat additional sequence of CD4 beyond the transmembrane domain of aminoacids 397 to 418 can be included in a CAR, if desired. It is furtherunderstood that sequences of CD4 that are shorter or longer than aspecific delineated domain can be included in a CAR, if desired.

CD8. Cluster of differentiation 8 (CD8) is a transmembrane glycoproteinthat serves as a co-receptor for the T cell receptor (TCR). CD8 binds toa major histocompatibility complex (MEC) molecule and is specific forthe class I MEC protein. In some embodiments, a CAR can comprise atransmembrane domain derived from CD8. A CD8 polypeptide can have anamino acid sequence corresponding to the sequence having GenBank No.NP_001139345.1 (GI:225007536), as provided below, or fragments thereof.See GenBank NP_001139345.1 for reference to domains within CD8, forexample, signal peptide, amino acids 1 to 21; extracellular domain,amino acids 22 to 182; transmembrane domain amino acids, 183 to 203;intracellular domain, amino acids 204 to 235. In some embodiments, a CARcan comprise a hinge domain derived from CD8. In some embodiments, thehinge domain can comprise amino acids 137 to 182 of the CD8 polypeptideprovided below. In some embodiments, a CAR can comprise a transmembranedomain derived from CD8. In some embodiments, the transmembrane domainof the CAR comprises the transmembrane region of CD8 (e.g., amino acids183 to 203 of the sequence below), or a fragment thereof. In anotherembodiment, a CAR can comprise amino acids 137 to 203 of the CD8polypeptide provided below. In yet another embodiment, a CAR cancomprise amino acids 137 to 209 of the CD8 polypeptide provided below.It is understood that additional sequence of CD8 beyond the hinge domainof amino acids 137 to 182 and the transmembrane domain of amino acids183 to 203 can be included in a CAR, if desired. It is furtherunderstood that sequences of CD8 that are shorter or longer than aspecific delineated domain can be included in a CAR, if desired.

(SEQ ID NO: 24) 1 MALPVTALLL PLALLLHAAR PSQFRVSPLDRTWNLGETVE LKCQVLLSNP TSGCSWLFQP 61 RGAAASPTFL LYLSQNKPKA AEGLDTQRFSGKRLGDTFVL TLSDFRRENE GYYFCSALSN 121 SIMYFSHFVP VFLPAKPTTT PAPRPPTPAPTIASQPLSLR PEACRPAAGG AVHTRGLDFA 181 CDIYIWAPLA GTCGVLLLSL VITLYCNHRNRRRVCKCPRP VVKSGDKPSL SARYV

As such, for exemplary purposes, a CAR disclosed herein can comprise,from N-terminus to the C-terminus, an anti-BCMA antibody orantigen-binding fragment (e.g., scFvs disclosed herein), a hinge (e.g.,CD8 hinge or CD28 hinge), a transmembrane region (e.g., CD8transmembrane region or CD28 transmembrane region), a costimulatorydomain (e.g., the intracellular domain of 4-1BB, CD28, or both), and asignaling domain (e.g., the T cell signaling domain of CD3).

5.4 Polynucleotides and Vectors

Also provided herein are polynucleotides that encode a polypeptide(e.g., an anti-BCMA antibody or antigen-binding fragment or a CAR thatspecifically binds BCMA, or a fusion protein) described herein. The term“polynucleotide that encode a polypeptide” encompasses a polynucleotidewhich includes only coding sequences for the polypeptide as well as apolynucleotide which includes additional coding and/or non-codingsequences. The polynucleotides of the disclosure can be in the form ofRNA or in the form of DNA. DNA can be cDNA, genomic DNA, or syntheticDNA, and can be double-stranded or single-stranded. Single stranded DNAcan be the coding strand or non-coding (anti-sense) strand. Thepolynucleotides of the disclosure can be mRNA.

Expressly contemplated herein are polynucleotides encode any anti-BCMAantibody or antigen-binding fragment disclosed herein. For illustrativepurposes, in some embodiments, the polynucleotides provided hereinencode an anti-BCMA antibody or antigen-binding fragment comprising (a)a VL comprising (1) a VL CDR1 having an amino acid sequence of SEQ IDNO:1; (2) a VL CDR2 having an amino acid sequence of SEQ ID NO:2; and(3) a VL CDR3 having an amino acid sequence of SEQ ID NO:3; or a variantthereof having up to about 5 amino acid substitutions, additions, and/ordeletions in the VL CDRs; and/or (b) a VH comprising (1) a VH CDR1having an amino acid sequence of SEQ ID NO:4; (2) a VH CDR2 having anamino acid sequence of SEQ ID NO:5; and (3) a VH CDR3 having an aminoacid sequence of SEQ ID NO:6; or a variant thereof having up to about 5amino acid substitutions, additions, and/or deletions in the VH CDRs. Insome embodiments, the polynucleotides provided herein encode ananti-BCMA antibody or antigen-binding fragment comprising (a) a VLhaving at least 85%, at least 90%, at least 95%, at least 98%, or 100%sequence identity to an amino acid sequence of SEQ ID NO:7; and/or (b) aVH having at least 85%, at least 90%, at least 95%, at least 98%, or100% sequence identity to an amino acid sequence of SEQ ID NO:8. Thepolynucleotides can be in the form of DNA. The polynucleotides can be inthe form of mRNA.

In some embodiments, the polynucleotides provided herein encode ananti-BCMA antibody or antigen-binding fragment disclosed hereincomprising a VL and a VH, wherein the VL comprises VL CDR1, CDR2 andCDR3 and the VH comprises VH CDR1, CDR2 and CDR3, and wherein the VLCDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2 and VH CDR3 have the amino acidsequences of SEQ ID NO:1, 2, 3, 4, 5, and 6, respectively, or a variantthereof having up to about 5 amino acid substitutions, additions, and/ordeletions in the VL CDRs. The polynucleotides can be in the form of DNA.The polynucleotides can be in the form of mRNA.

In some embodiments, the polynucleotides provided herein encode ananti-BCMA antibody or antigen-binding fragment disclosed hereincomprising a VL and a VH, wherein the VL and VH have the amino acidsequences of SEQ ID NO:7 and 8, respectively. The polynucleotides can bein the form of DNA. The polynucleotides can be in the form of mRNA.

In some embodiments, the VL and VH are connected by a linker. The linkercan be a flexible linker or a rigid linker. In some embodiments, thelinker has the amino acid sequence of (GGGGS)n, n=1, 2, 3, 4, or 5. Forexample, the linker may have the amino acid sequence of GGGGS (SEQ IDNO: 17). In some embodiments, the linker has the amino acid sequence of(EAAAK)n, n=1, 2, 3, 4, or 5. For example, the linker may have the aminoacid sequence of EAAAK (SEQ ID NO: 18). In some embodiments, the linkerhas the amino acid sequence of (PA)nP, n=1, 2, 3, 4, or 5. For example,the linker may have the amino acid sequence of PAP (SEQ ID NO: 19). Insome embodiments, the linker has the amino acid sequence ofGGGGSGGGGSGGGGS (SEQ ID NO: 20).

In some embodiments, the polynucleotides provided herein encode ananti-BCMA antibody or antigen-binding fragment disclosed hereincomprising a VL having at least 85%, at least 90%, at least 95%, atleast 98%, or 100% sequence identity to an amino acid sequence of SEQ IDNO:7.

In some embodiments, the polynucleotides provided herein have anucleotide sequence at least 80%, at least 85%, at least 90%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identical to a nucleotide sequence of SEQ ID NO:9. Also provided is apolynucleotide that hybridizes to a polynucleotide having a nucleotidesequence of SEQ ID NO:9. In some embodiments, the hybridization is underconditions of high stringency as is known to those skilled in the art.The polynucleotides can be in the form of DNA. The polynucleotides canbe in the form of mRNA.

In some embodiments, the polynucleotides provided herein encode ananti-BCMA antibody or antigen-binding fragment disclosed hereincomprising a VH having at least 85%, at least 90%, at least 95%, atleast 98%, or 100% sequence identity to an amino acid sequence of SEQ IDNO:8. In some embodiments, the polynucleotides provided herein encode ananti-BCMA antibody or antigen-binding fragment disclosed hereincomprising a VH having an amino acid sequence of SEQ ID NO:8.

In some embodiments, the polynucleotides provided herein have anucleotide sequence at least 80%, at least 85%, at least 90%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identical to a nucleotide sequence of SEQ ID NO:10. Also provided is apolynucleotide that hybridizes to a polynucleotide having a nucleotidesequence of SEQ ID NO:10. In some embodiments, the hybridization isunder conditions of high stringency as is known to those skilled in theart. The polynucleotides can be in the form of DNA. The polynucleotidescan be in the form of mRNA.

The present disclosure also provides variants of the polynucleotidesdescribed herein, wherein the variants encode, for example, fragments,analogs, and/or derivatives of an anti-BCMA antibody or antigen-bindingfragment disclosed herein. In some embodiments, the present disclosureprovides a polynucleotide having a nucleotide sequence at least about80% identical, at least about 85% identical, at least about 90%identical, at least about 95% identical, at least about 96% identical,at least about 97% identical, at least about 98% identical, or at leastabout 99% identical to a polynucleotide sequence encoding an anti-BCMAantibody or antigen-binding fragment described herein.

In some embodiments, the polynucleotides provided herein encode ananti-BCMA antibody or antigen-binding fragment that is the scFvdesignated as BCMA31. In some embodiments, the polynucleotides providedherein encode an anti-BCMA antibody or antigen-binding fragment havingthe amino acid sequence of SEQ ID NO:11.

Provided herein are also polynucleotides that encode the TCRs disclosedherein. In some embodiments, provided herein are polynucleotides thatencode a TCR α chain that comprises an anti-BCMA antibody orantigen-binding fragment described herein. In some embodiments, providedherein are polynucleotides that encode a TCR β chain that comprises ananti-BCMA antibody or antigen-binding fragment described herein. In someembodiments, provided herein are polynucleotides that encode a TCR γchain that comprises an anti-BCMA antibody or antigen-binding fragmentdescribed herein. In some embodiments, provided herein arepolynucleotides that encode a TCR δ chain that comprises an anti-BCMAantibody or antigen-binding fragment described herein. Thepolynucleotides can be in the form of DNA. The polynucleotides can be inthe form of mRNA.

Provided herein are also polynucleotides that encode the CARs disclosedherein. In some embodiments, provided herein are polynucleotidesencoding CARs that specifically binds BCMA, comprising, from N-terminusto C-terminus: (a) a BCMA binding domain comprising an anti-BCMAantibody or antigen-binding fragment provided herein, (b) atransmembrane domain, and (c) a cytoplasmic domain. The transmembraneand cytoplasmic domains can be any transmembrane and cytoplasmic domainsdisclosed herein. For illustrative purposes, provided herein are, forexample, polynucleotides that encode the CARs that specifically bindsBCMA, comprising, from N-terminus to C-terminus: (a) a BCMA bindingdomain comprising an anti-BCMA scFv provided herein, (b) a transmembranedomain comprising the CD28 transmembrane region, and (c) a cytoplasmicdomain comprising a CD3ζ signaling domain and a 4-1BB co-stimulatorydomain. The polynucleotides can be in the form of DNA. Thepolynucleotides can be in the form of mRNA.

In some embodiments, the polynucleotides provided herein encode ananti-BCMA CAR having at least 85%, at least 90%, at least 95%, at least98%, or 100% sequence identity to an amino acid sequence of SEQ IDNO:15. In some embodiments, the polynucleotides provided herein encodean anti-BCMA CAR having an amino acid sequence of SEQ ID NO:15.

In some embodiments, the polynucleotides provided herein have anucleotide sequence at least 80%, at least 85%, at least 90%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identical to a nucleotide sequence of SEQ ID NO:16. Also provided is apolynucleotide that hybridizes to a polynucleotide encoding an aminoacid sequence SEQ ID NO:16. In some embodiments, the hybridization isunder conditions of high stringency as is known to those skilled in theart.

As used herein, the phrase “a polynucleotide having a nucleotidesequence at least about 95% identical to a polynucleotide sequence”means that the nucleotide sequence of the polynucleotide is identical toa reference sequence except that the polynucleotide sequence can includeup to five point mutations per each 100 nucleotides of the referencenucleotide sequence. In other words, to obtain a polynucleotide having anucleotide sequence at least 95% identical to a reference nucleotidesequence, up to 5% of the nucleotides in the reference sequence can bedeleted or substituted with another nucleotide, or a number ofnucleotides up to 5% of the total nucleotides in the reference sequencecan be inserted into the reference sequence. These mutations of thereference sequence can occur at the 5′ or 3′ terminal positions of thereference nucleotide sequence or anywhere between those terminalpositions, interspersed either individually among nucleotides in thereference sequence or in one or more contiguous groups within thereference sequence.

The polynucleotide variants can contain alterations in the codingregions, non-coding regions, or both. In some embodiments, apolynucleotide variant contains alterations which produce silentsubstitutions, additions, or deletions, but does not alter theproperties or activities of the encoded polypeptide. In someembodiments, a polynucleotide variant comprises silent substitutionsthat results in no change to the amino acid sequence of the polypeptide(due to the degeneracy of the genetic code). Polynucleotide variants canbe produced for a variety of reasons, for example, to optimize codonexpression for a particular host (e.g., change codons in the human mRNAto those preferred by a bacterial host such as E. coli). In someembodiments, a polynucleotide variant comprises at least one silentmutation in a non-coding or a coding region of the sequence.

In some embodiments, a polynucleotide variant is produced to modulate oralter expression (or expression levels) of the encoded polypeptide. Insome embodiments, a polynucleotide variant is produced to increaseexpression of the encoded polypeptide. In some embodiments, apolynucleotide variant is produced to decrease expression of the encodedpolypeptide. In some embodiments, a polynucleotide variant has increasedexpression of the encoded polypeptide as compared to a parentalpolynucleotide sequence. In some embodiments, a polynucleotide varianthas decreased expression of the encoded polypeptide as compared to aparental polynucleotide sequence.

In some embodiments, a polynucleotide comprises the coding sequence fora polypeptide (e.g., a CAR or an antibody) fused in the same readingframe to a polynucleotide which aids in expression and secretion of apolypeptide from a host cell (e.g., a leader sequence which functions asa secretory sequence for controlling transport of a polypeptide). Thepolypeptide can have the leader sequence cleaved by the host cell toform a “mature” form of the polypeptide.

In some embodiments, a polynucleotide comprises the coding sequence fora polypeptide (e.g., a CAR or an antibody) fused in the same readingframe to a marker or tag sequence. For example, in some embodiments, amarker sequence is a hexa-histidine tag (HIS-tag) that allows forefficient purification of the polypeptide fused to the marker. In someembodiments, a marker sequence is a hemagglutinin (HA) tag derived fromthe influenza hemagglutinin protein when a mammalian host (e.g., COS-7cells) is used. In some embodiments, the marker sequence is a FLAG™ tag.In some embodiments, a marker can be used in conjunction with othermarkers or tags.

In some embodiments, a polynucleotide is isolated. In some embodiments,a polynucleotide is substantially pure.

Vectors and cells comprising the polynucleotides described herein arealso provided. In some embodiments, provided herein are vectorscomprising a polynucleotide provided herein. The vectors can beexpression vectors. In some embodiments, vectors provided hereincomprise a polynucleotide encoding an anti-BCMA antibody orantigen-binding fragment described herein. In some embodiments, vectorsprovided herein comprise a polynucleotide encoding a polypeptide that ispart of an anti-BCMA antibody or antigen-binding fragment describedherein. In some embodiments, vectors provided herein comprise apolynucleotide encoding a CAR or TCR described herein. In someembodiments, vectors provided herein comprise a polynucleotide encodinga polypeptide that is part of a CAR or TCR described herein.

In some embodiments, provided herein are recombinant expression vectors,which can be used to amplify and express a polynucleotide encoding aCAR/TCR described herein that specifically binds BCMA or an anti-BCMAantibody or antigen-binding fragment described herein. For example, arecombinant expression vector can be a replicable DNA construct thatincludes synthetic or cDNA-derived DNA fragments encoding a CAR/TCR or apolypeptide chain of an anti-BCMA antibody, operatively linked tosuitable transcriptional and/or translational regulatory elementsderived from mammalian, microbial, viral or insect genes. In someembodiments, a viral vector is used. DNA regions are “operativelylinked” when they are functionally related to each other. For example, apromoter is operatively linked to a coding sequence if it controls thetranscription of the sequence; or a ribosome binding site is operativelylinked to a coding sequence if it is positioned so as to permittranslation. In some embodiments, structural elements intended for usein certain expression systems include a leader sequence enablingextracellular secretion of translated protein by a host cell. In someembodiments, in situations where recombinant protein is expressedwithout a leader or transport sequence, a polypeptide can include anN-terminal methionine residue.

A wide variety of expression host/vector combinations can be employed.Useful expression vectors for eukaryotic hosts include, for example,vectors comprising expression control sequences from SV40, bovinepapilloma virus, adenovirus, and cytomegalovirus. Useful expressionvectors for bacterial hosts include known bacterial plasmids, such asplasmids from E. coli, including pCR1, pBR322, pMB9 and theirderivatives, and wider host range plasmids, such as M13 and otherfilamentous single-stranded DNA phages.

In some embodiments, a CAR/TCR described herein or an anti-BCMA antibodyor antigen-binding fragment described herein is expressed from one ormore vectors. Suitable host cells for expression include prokaryotes,yeast cells, insect cells, or higher eukaryotic cells under the controlof appropriate promoters. Appropriate cloning and expression vectors foruse with bacterial, fungal, yeast, and mammalian cellular hosts, as wellas methods of protein production, including antibody production arewell-known in the art.

Examples of suitable mammalian host cell lines include, but are notlimited to, COS-7 (monkey kidney-derived), L-929 (murinefibroblast-derived), C127 (murine mammary tumor-derived), 3T3 (murinefibroblast-derived), CHO (Chinese hamster ovary-derived), HeLa (humancervical cancer-derived), BHK (hamster kidney fibroblast-derived),HEK-293 (human embryonic kidney-derived) cell lines and variantsthereof. Mammalian expression vectors can comprise non-transcribedelements such as an origin of replication, a suitable promoter andenhancer linked to the gene to be expressed, and other 5′ or 3′ flankingnon-transcribed sequences, and 5′ or 3′ non-translated sequences, suchas necessary ribosome binding sites, a polyadenylation site, splicedonor and acceptor sites, and transcriptional termination sequences.Expression of recombinant proteins in insect cell culture systems (e.g.,baculovirus) also offers a robust method for producing correctly foldedand biologically functional proteins. Baculovirus systems for productionof heterologous proteins in insect cells are well-known to those ofskill in the art.

The present disclosure also provides host cells comprising thepolypeptides described herein, polynucleotides encoding polypeptidesdescribed herein, or vectors comprising such polynucleotides. In someembodiments, provided herein are host cells comprising a vectorcomprising a polynucleotide disclosed herein. In some embodiments, hostcells provided herein comprise a vector comprising a polynucleotideencoding an anti-BCMA antibody or antigen-binding fragment describedherein. In some embodiments, host cells provided herein comprise avector comprising a polynucleotide encoding a polypeptide that is partof an anti-BCMA antibody or antigen-binding fragment described herein.In some embodiments, host cells provided herein comprise apolynucleotide encoding an anti-BCMA antibody or antigen-bindingfragment described herein. In some embodiments, the cells produce theanti-BCMA antibodies or antigen-binding fragments described herein. Insome embodiments, host cells provided herein comprise a vectorcomprising a polynucleotide encoding a CAR or TCR described herein. Insome embodiments, host cells provided herein comprise a vectorcomprising a polynucleotide molecule encoding a polypeptide that is partof a CAR or TCR described herein. In some embodiments, host cellsprovided herein comprise a polynucleotide encoding a CAR or TCRdescribed herein. In some embodiments, the host cells produce the BCMACARs or TCR described herein.

5.5 Cells

Provided herein are cells comprising the polynucleotides disclosedherein. In some embodiments, provided herein are cells comprising apolynucleotide that encodes a polypeptide disclosed herein. In someembodiments, provided herein are cells comprising a vector having apolynucleotide disclosed herein. In some embodiments, provided hereinare cells recombinantly expressing a polypeptide disclosed herein. Thepolypeptide can be an anti-BCMA antibody or antigen-binding fragment.The polypeptide can be BCMA CAR. The polypeptide can be a BCMA TCR.

In some embodiments, cells provided herein are immune effector cells. Insome embodiments, the immune effector cells are selected from the groupconsisting of T cells, B cell, natural killer (NK) cells, NKT cells,macrophages, granulocytes, neutrophils, eosinophils, mast cells, andbasophils. In some embodiments, the immune effector cells providedherein are selected from the group consisting of T cells, NK cells, NKTcells, macrophages, neutrophils, and granulocytes. In some embodiments,the immune effector cell provided herein is a T cell. In someembodiments, the immune effector cell provided herein is an NK cell. Insome embodiments, the immune effector cell provided herein is an NKTcell. In some embodiments, the immune effector cell provided herein is amacrophage. In some embodiments, the immune effector cell providedherein is a neutrophil. In some embodiments, the immune effector cellprovided herein is a granulocyte.

In some embodiments, the immune effector cells provided herein can begenetically engineered. In some embodiments, the genetically engineeredimmune effector cells provided herein are isolated. In some embodiments,the genetically engineered immune effector cells provided herein aresubstantially pure.

As such, in some embodiments, provided herein are immune effector cellsrecombinantly expressing a polypeptide (e.g., an antibody or a CAR)disclosed herein. Provided herein are also immune effector cells (e.g.,T cells) comprising a polynucleotide encoding a polypeptide (e.g., anantibody or a CAR) disclosed herein, or a vector having a polynucleotidedisclosed herein. In some embodiments, provided herein are immuneeffector cells (e.g., T cells) comprising a polynucleotide that encodesan anti-BCMA antibody or antigen-binding fragment disclosed herein. Insome embodiments, provided herein are immune effector cells (e.g., Tcells) recombinantly expressing an anti-BCMA antibody or antigen-bindingfragment disclosed herein. In some embodiments, provided herein areimmune effector cells comprising a polynucleotide that encodes a BCMACAR disclosed herein. In some embodiments, provided herein are immuneeffector cells (e.g., T cells) recombinantly expressing a BCMA CARdisclosed herein (e.g., BCMA CART cell). In some embodiments, providedherein are immune effector cells comprising a polynucleotide thatencodes a BCMA TCR disclosed herein. In some embodiments, providedherein are immune effector cells (e.g., T cells) recombinantlyexpressing a BCMA TCR disclosed herein (e.g., BCMA TCRT cell).

In some embodiments, the immune effector cell provided herein is a Tcell. The T cell can be a cytotoxic T cell, a helper T cell, or a gammadelta T, a CD4+/CD8+ double positive T cell, a CD4+ T cell, a CD8+ Tcell, a CD4/CD8 double negative T cell, a CD3+ T cell, a naive T cell,an effector T cell, a cytotoxic T cell, a helper T cell, a memory Tcell, a regulator T cell, a Th0 cell, a Th1 cell, a Th2 cell, a Th3(Treg) cell, a Th9 cell, a Th17 cell, a Thαβ helper cell, a Tfh cell, astem memory TSCM cell, a central memory TCM cell, an effector memory TEMcell, an effector memory TEMRA cell, or a gamma delta T cell. In someembodiments, the T cell is a cytotoxic T cell. In some embodiments, theT cell is genetically engineered. In some embodiments, the T cellsprovided herein are isolated. In some embodiments, the T cells providedherein are substantially pure.

In some embodiments, genetically engineered cells provided herein arederived from cells isolated from a subject. As used herein, agenetically engineered cell that is “derived from” a source cell meansthat the genetically engineered cell is obtained by taking the sourcecell and genetically manipulating the source cell. The source cell canbe from a natural source. For example, the source cell can be a primarycell isolated from a subject. The subject can be an animal or a human.The source cell can also be a cell that has undergone passages orgenetically manipulation in vitro.

In some embodiments, genetically engineered cells provided herein arederived from cells isolated from a human. Immune effector cells (e.g., Tcells) can be obtained from many sources, including peripheral bloodmononuclear cells, bone marrow, lymph node tissue, cord blood, thymustissue, tissue from a site of infection, ascites, pleural effusion,spleen tissue, and tumors. In certain embodiments, T cell linesavailable in the art can be used. In some embodiments, geneticallyengineered cells provided herein are derived from cells isolated fromperipheral blood. In some embodiments, genetically engineered cellsprovided herein are derived from cells isolated from bone marrow. Insome embodiments, genetically engineered cells provided herein arederived from cells isolated from peripheral blood mononuclear cells(PBMC).

In some embodiments, genetically engineered cells provided herein arederived from cells differentiated in vitro from a stem or progenitorcell. In some embodiments, the stem or progenitor cell is selected fromthe group consisting of a T cell progenitor cell, a hematopoietic stemand progenitor cell, a hematopoietic multipotent progenitor cell, anembryonic stem cell, and an induced pluripotent cell. In someembodiments, genetically engineered cells provided herein are derivedfrom cells differentiated in vitro from a T cell progenitor cell. Insome embodiments, genetically engineered cells provided herein arederived from cells differentiated in vitro from a hematopoietic stem andprogenitor cell. In some embodiments, genetically engineered cellsprovided herein are derived from cells differentiated in vitro from ahematopoietic multipotent progenitor cell. In some embodiments,genetically engineered cells provided herein are derived from cellsdifferentiated in vitro from an embryonic stem cell. In someembodiments, genetically engineered cells provided herein are derivedfrom cells differentiated in vitro from an induced pluripotent cell.

In some embodiments, provided herein are a population of cellscomprising a cell disclosed herein. The cells disclosed herein cancomprise a polynucleotide that encodes a polypeptide disclosed herein orrecombinantly express a polypeptide disclosed herein. The polypeptidecan be an anti-BCMA antibody or antigen-binding fragment, a BCMA CAR, ora BCMA TCR.

The population of cells can be a homogenous population of cells. Thepopulation of cells can be a heterogeneous population of cells. In someembodiments, the population of cells can be a heterogeneous populationof cells comprising any combination of the cells disclosed herein. Insome embodiments, the population of cells are derived from peripheralblood mononuclear cells (PBMC), peripheral blood leukocytes (PBL), tumorinfiltrating lymphocytes (TIL), cytokine-induced killer cells (CIK),lymphokine-activated killer cells (LAK), or marrow infiltratelymphocytes (MILs). In some embodiments, the population of cellsprovided herein are derived from PBMC. In some embodiments, thepopulation of cells provided herein are derived from PBL. In someembodiments, the population of cells provided herein are derived fromTIL. In some embodiments, the population of cells provided herein arederived from CIK. In some embodiments, the population of cells providedherein are derived from LAK. In some embodiments, the population ofcells provided herein are derived from MILS. The population of cells canbe genetically engineered to recombinantly expressing a polypeptide(e.g., an antibody or a CAR) disclosed herein. In some embodiments,provided herein are population of cells comprising a polynucleotideencoding a polypeptide (e.g., an antibody or a CAR) disclosed herein, ora vector having a polynucleotide disclosed herein. In some embodiments,provided herein are population of cells comprising a polynucleotide thatencodes an anti-BCMA antibody or antigen-binding fragment disclosedherein. In some embodiments, provided herein are population of cellsrecombinantly expressing an anti-BCMA antibody or antigen-bindingfragment disclosed herein. In some embodiments, provided herein arepopulation of cells comprising a polynucleotide that encodes a BCMA CARdisclosed herein. In some embodiments, provided herein are population ofcells recombinantly expressing a BCMA CAR disclosed herein (e.g., BCMACART cell). In some embodiments, provided herein are population of cellscomprising a polynucleotide that encodes a BCMA TCR disclosed herein. Insome embodiments, provided herein are population of cells recombinantlyexpressing a BCMA TCR disclosed herein (e.g., BCMA TCRT cell).

5.6 Pharmaceutical Compositions

Provided herein are also pharmaceutical compositions comprising theanti-BCMA antibodies or antigen-binding fragments disclosed herein.Provided herein are also pharmaceutical compositions comprising thegenetically engineered immune effector cells disclosed herein. In someembodiments, the pharmaceutical composition comprises a therapeuticallyeffective amount of the anti-BCMA antibodies or antigen-bindingfragments disclosed herein and a pharmaceutically acceptable carrier. Insome embodiments, the pharmaceutical composition comprises atherapeutically effective amount of genetically engineered cellsdisclosed herein and a pharmaceutically acceptable carrier. In someembodiments, the pharmaceutical compositions are useful inimmunotherapy. In some embodiments, the pharmaceutical compositions areuseful in immuno-oncology. In some embodiments, the pharmaceuticalcompositions are useful in inhibiting tumor growth in a subject (e.g., ahuman patient). In some embodiments, the pharmaceutical compositions areuseful in treating cancer in a subject (e.g., a human patient).

In some embodiments, the pharmaceutical compositions provided hereincomprise anti-BCMA antibodies or antigen-binding fragments providedherein. The anti-BCMA antibodies or antigen-binding fragments can bepresent at various concentrations. In some embodiments, thepharmaceutical compositions provided herein comprise soluble anti-BCMAantibodies or antigen-binding fragments provided herein at 1-1000 mg/ml.In some embodiments, the pharmaceutical compositions comprise solubleanti-BCMA antibodies or antigen-binding fragments provided herein at10-500 mg/ml, 10-400 mg/ml, 10-300 mg/ml, 10-200 mg/ml, 10-100 mg/ml,20-100 mg/ml, or 50-100 mg/ml. In some embodiments, the pharmaceuticalcompositions provided herein comprise anti-BCMA antibodies orantigen-binding fragments provided herein at about 10 mg/ml, about 20mg/ml, about 30 mg/ml, about 40 mg/ml, about 50 mg/ml, about 60 mg/ml,about 70 mg/ml, about 80 mg/ml, about 90 mg/ml, about 100 mg/ml, about120 mg/ml, about 150 mg/ml, about 180 mg/ml, about 200 mg/ml, about 300mg/ml, about 500 mg/ml, about 800 mg/ml, or about 1000 mg/ml.

The pharmaceutical compositions comprising genetically engineered immuneeffector cells (e.g., T cells) disclosed herein can comprise a purifiedpopulation of cells. Those skilled in the art can readily determine thepercentage of cells in a cell population using various well-knownmethods, as described herein. The ranges of purity in cell populationscomprising genetically engineered cells provided herein can be fromabout 20% to about 25%, from about 25% to about 30%, from about 30% toabout 35%, from about 35% to about 40%, from about 40% to about 45%,from about 45% to about 50%, from about 55% to about 60%, from about 65%to about 70%, from about 70% to about 75%, from about 75% to about 80%,from about 80% to about 85%; from about 85% to about 90%, from about 90%to about 95%, or from about 95 to about 100%. In some embodiments, theranges of purity in cell populations comprising immune effector cellsprovided herein can be from about 20% to about 30%, from about 20% toabout 50%, from about 20% to about 80%, from about 20% to about 100%,from about 50% to about 80%, or from about 50% to about 100%. Dosagescan be readily adjusted by those skilled in the art; for example, adecrease in purity may require an increase in dosage.

Provided herein are also kits for preparation of pharmaceuticalcompositions having the anti-BCMA antibodies or antigen-bindingfragments disclosed herein. In some embodiments, the kit comprises theanti-BCMA antibodies or antigen-binding fragments disclosed herein and apharmaceutically acceptable carrier in one or more containers. Inanother embodiment, the kits can comprise anti-BCMA antibodies orantigen-binding fragments disclosed herein for administration to asubject. In specific embodiments, the kits comprise instructionsregarding the preparation and/or administration of the anti-BCMAantibodies or antigen-binding fragments.

Provided herein are also kits for preparation of immune effector cells(e.g., T cells) disclosed herein. In some embodiments, the kits compriseone or more vectors for generating a genetically engineered cell, suchas a T cell, that expresses the anti-BCMA antibodies or antigen-bindingfragments disclosed herein. The kits can be used to generate geneticallyengineered immune effector cells (e.g., T cells) from autologous ornon-autologous cells to be administered to a compatible subject. Inanother embodiment, the kits can comprise immune effector cellsdisclosed herein for administration to a subject. In specificembodiments, the kits comprise the immune effector cells disclosedherein in one or more containers. In specific embodiments, the kitscomprise instructions regarding the preparation and/or administration ofthe immune effector cells.

In some embodiments, provided herein is a pharmaceutical compositioncomprising anti-BCMA antibodies or antigen-binding fragments or cellsprovided herein wherein the composition is suitable for localadministration. In some embodiments, local administration comprisesintratumoral injection, peritumoral injection, juxtatumoral injection,intralesional injection and/or injection into a tumor draining lymphnode, or essentially any tumor-targeted injection where the antitumoragent is expected to leak into primary lymph nodes adjacent to targetedsolid tumor.

Pharmaceutically acceptable carriers that can be used in compositionsprovided herein include any and all solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents, and the like that are physiologically compatible. Insome embodiments, the carrier is suitable for intravenous,intramuscular, subcutaneous, parenteral, spinal or epidermaladministration (e.g., by injection or infusion). Depending on the routeof administration, the active ingredient (i.e., anti-BCMA antibodies orantigen-binding fragments or immune effector cells provided herein), canbe coated in a material to protect the active ingredient from the actionof acids and other natural conditions that can inactivate the activeingredient.

Provided herein are also pharmaceutical compositions or formulationsthat improve the stability of the anti-BCMA antibodies orantigen-binding fragments to allow for their long-term storage. In someembodiments, the pharmaceutical composition or formulation disclosedherein comprises: (a) anti-BCMA antibodies or antigen-binding fragmentsdisclosed herein; (b) a buffering agent; (c) a stabilizing agent; (d) asalt; (e) a bulking agent; and/or (f) a surfactant. In some embodiments,the pharmaceutical composition or formulation is stable for at least 1month, at least 2 months, at least 3 months, at least 6 months, at least1 year, at least 2 years, at least 3 years, at least 5 years or more. Insome embodiments, the pharmaceutical composition or formulation isstable when stored at 4° C., 25° C., or 40° C.

Buffering agents useful in the pharmaceutical compositions orformulations disclosed herein can be a weak acid or base used tomaintain the acidity (pH) of a solution near a chosen value after theaddition of another acid or base. Suitable buffering agents can maximizethe stability of the pharmaceutical formulations by maintaining pHcontrol of the formulation. Suitable buffering agents can also ensurephysiological compatibility or optimize solubility. Rheology, viscosityand other properties can also depend on the pH of the formulation.Common buffering agents include, but are not limited to, histidine,citrate, succinate, acetate and phosphate. In some embodiments, abuffering agent comprises histidine (e.g., L-histidine) with isotonicityagents and potentially pH adjustment with an acid or a base known in theart. In certain embodiments, the buffering agent is L-histidine. Incertain embodiments, the pH of the formulation is maintained betweenabout 2 and about 10, or between about 4 and about 8.

Stabilizing agents are added to a pharmaceutical product to stabilizethat product. Such agents can stabilize proteins in different ways.Common stabilizing agents include, but are not limited to, amino acidssuch as glycine, alanine, lysine, arginine, or threonine, carbohydratessuch as glucose, sucrose, trehalose, rafftnose, or maltose, polyols suchas glycerol, mannitol, sorbitol, cyclodextrins or destrans of any kindand molecular weight, or PEG. In some embodiments, the stabilizing agentis chosen to maximize the stability of FIX polypeptide in lyophilizedpreparations. In certain embodiments, the stabilizing agent is sucroseand/or arginine.

Bulking agents can be added to a pharmaceutical composition orformulation to add volume and mass to the product, thereby facilitatingprecise metering and handling thereof. Common bulking agents include,but are not limited to, lactose, sucrose, glucose, mannitol, sorbitol,calcium carbonate, or magnesium stearate.

Surfactants are amphipathic substances with lyophilic and lyophobicgroups. A surfactant can be anionic, cationic, zwitterionic, ornonionic. Examples of nonionic surfactants include, but are not limitedto, alkyl ethoxylate, nonylphenol ethoxylate, amine ethoxylate,polyethylene oxide, polypropylene oxide, fatty alcohols such as cetylalcohol or oleyl alcohol, cocamide MEA, cocamide DEA, polysorbates, ordodecyl dimethylamine oxide. In some embodiments, the surfactant ispolysorbate 20 or polysorbate 80.

The pharmaceutical compositions disclosed herein can further compriseone or more of a buffer system, a preservative, a tonicity agent, achelating agent, a stabilizer and/or a surfactant, as well as variouscombinations thereof. The use of preservatives, isotonic agents,chelating agents, stabilizers and surfactants in pharmaceuticalcompositions is well-known to the skilled person. Reference may be madeto Remington: The Science and Practice of Pharmacy, 19^(th) edition,1995.

In some embodiments, the pharmaceutical composition is an aqueousformulation. Such a formulation is typically a solution or a suspension,but can also include colloids, dispersions, emulsions, and multi-phasematerials. The term “aqueous formulation” is defined as a formulationcomprising at least 50% w/w water. Likewise, the term “aqueous solution”is defined as a solution comprising at least 50% w/w water, and the term“aqueous suspension” is defined as a suspension comprising at least 50%w/w water.

In some embodiments, the pharmaceutical compositions disclosed hereinare freeze-dried, to which the physician or the patient adds solventsand/or diluents prior to use.

Pharmaceutical compositions disclosed herein can also include apharmaceutically acceptable antioxidant. Examples of pharmaceuticallyacceptable antioxidants include: (1) water soluble antioxidants, such asascorbic acid, cysteine hydrochloride, sodium bisulfate, sodiummetabisulfite, sodium sulfite and the like; (2) oil-solubleantioxidants, such as ascorbyl palmitate, butylated hydroxyanisole(BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate,alpha-tocopherol, and the like; and (3) metal chelating agents, such ascitric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaricacid, phosphoric acid, and the like.

Examples of suitable aqueous and nonaqueous carriers that can beemployed in the pharmaceutical compositions or formulations describedherein include water, ethanol, polyols (such as glycerol, propyleneglycol, polyethylene glycol, and the like), and suitable mixturesthereof, vegetable oils, such as olive oil, and injectable organicesters, such as ethyl oleate. Proper fluidity can be maintained, forexample, by the use of coating materials, such as lecithin, by themaintenance of the required particle size in the case of dispersions,and by the use of surfactants.

These compositions can also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofpresence of microorganisms can be ensured both by sterilizationprocedures, supra, and by the inclusion of various antibacterial andantifungal agents, for example, paraben, chlorobutanol, phenol sorbicacid, and the like. It can also be desirable to include isotonic agents,such as sugars, sodium chloride, and the like into the compositions. Inaddition, prolonged absorption of the injectable pharmaceutical form canbe brought about by the inclusion of agents which delay absorption suchas aluminum monostearate and gelatin.

Pharmaceutically acceptable carriers include sterile aqueous solutionsor dispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersion. The use of such media andagents for pharmaceutically active substances is known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the pharmaceutical compositionsdescribed herein is contemplated. A pharmaceutical composition orformulation can comprise a preservative or can be devoid of apreservative. Supplementary active compounds can be incorporated intothe compositions.

Pharmaceutical compositions or formulations typically must be sterileand stable under the conditions of manufacture and storage. Thecomposition can be formulated as a solution, microemulsion, liposome, orother ordered structure suitable to high drug concentration. The carriercan be a solvent or dispersion medium containing, for example, water,ethanol, polyol (for example, glycerol, propylene glycol, and liquidpolyethylene glycol, and the like), and suitable mixtures thereof. Theproper fluidity can be maintained, for example, by the use of a coatingsuch as lecithin, by the maintenance of the required particle size inthe case of dispersion and by the use of surfactants. In many cases, thecompositions can include isotonic agents, for example, sugars,polyalcohols such as mannitol, sorbitol, or sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent that delaysabsorption, for example, monostearate salts and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed bysterilization microfiltration. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated herein. In the case of sterile powders for the preparation ofsterile injectable solutions, some methods of preparation are vacuumdrying and freeze-drying (lyophilization) that yield a powder of theactive ingredient plus any additional desired ingredient from apreviously sterile-filtered solution thereof.

The amount of active ingredient which can be combined with a carriermaterial in the pharmaceutical compositions or formulations disclosedherein can vary. In some embodiments, the amount of active ingredientwhich can be combined with a carrier material is the amount thatproduces a therapeutic effect. Generally, out of one hundred percent,this amount will range from about 0.01 percent to about ninety-ninepercent of active ingredient, from about 0.1 percent to about 70percent, or from about 1 percent to about 30 percent of activeingredient in combination with a pharmaceutically acceptable carrier.

The pharmaceutical compositions disclosed herein can be prepared withcarriers that protect the active ingredient against rapid release, suchas a controlled release formulation, including implants, transdermalpatches, and microencapsulated delivery systems. Biodegradable,biocompatible polymers can be used, such as ethylene vinyl acetate,polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations arepatented or generally known to those skilled in the art. See. e.g.,Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson,ed., Marcel Dekker, Inc., New York, 1978.

In some embodiments, the anti-BCMA antibodies or antigen-bindingfragments or immune effector cells (e.g., T cells) described herein canbe formulated to ensure proper distribution in vivo. For example, theblood-brain barrier (BBB) excludes many highly hydrophilic compounds. Toensure that the activate ingredient described herein cross the BBB (ifdesired, e.g., for brain cancers), they can be formulated, for example,in liposomes. For methods of manufacturing liposomes, see, e.g., U.S.Pat. Nos. 4,522,811; 5,374,548; and 5,399,331. The liposomes cancomprise one or more moieties which are selectively transported intospecific cells or organs, thus enhance targeted drug delivery (see,e.g., V. V. Ranade (1989) J. Clin. Pharmacol. 29:685). Exemplarytargeting moieties include folate or biotin (see, e.g., U.S. Pat. No.5,416,016 to Low et al) mannosides (Umezawa et al, (1988) Biochem.Biophys. Res. Commun. 153: 1038); antibodies (P. G. Bloeman et al.(1995) FEBS Lett. 357: 140; M. Owais et al. (1995) Antimicrob. AgentsChemother. 39: 180); surfactant protein A receptor (Briscoe et al.(1995) Am. J. Physiol. 1233: 134); p120 (Schreier et al. (1994) J. Biol.Chem. 269:9090); see also K. Keinanen; M. L. Laukkanen (1994) FEBS Lett.346: 123; J. J. Killion; I. J. Fidler (1994) Immunomethods 4:273.

5.7 Methods and Uses

The present disclosure also provides methods of uses of the anti-BCMAantibodies or antigen-binding fragments, BCMA CARs, BCMA TCRs,polynucleotides encoding such anti-BCMA antibodies or antigen-bindingfragments and BCMA CARs/TCRs, vectors comprising such polynucleotides,BCMA CAR/TCR-expressing cells or pharmaceutical compositions having suchcells disclosed herein in treating cancer. Without being bound bytheory, the anti-BCMA antibodies or antigen-binding fragments and theBCMA CAR/TCR-expressing cells disclosed herein can specifically targetBCMA expressing cancer cells in vivo, thereby delivering theirtherapeutic effect of eliminating, lysing and/or killing cancer cells.In some embodiments, the methods include administering a therapeuticallyeffective amount of the anti-BCMA antibodies or antigen-bindingfragments disclosed herein to a subject in need thereof. In someembodiments, the methods include administering a therapeuticallyeffective amount of BCMA CAR-expressing immune effector cells disclosedherein to a subject in need thereof. In one embodiment, the methods caninclude administering a therapeutically effective amount of BCMA CARTsdisclosed herein to a subject in need thereof.

In some embodiments, provided herein are methods of treating tumor orcancer in a subject in need thereof, comprising administering to thesubject a therapeutically effective amount of the anti-BCMA antibodiesor antigen-binding fragments disclosed herein. In some embodiments,provided herein are uses of the anti-BCMA antibodies or antigen-bindingfragments disclosed herein in the treatment of tumor or cancer. In someembodiments, provided herein are uses of the anti-BCMA antibodies orantigen-binding fragments provided herein for the preparation of amedicament for the treatment of tumor or cancer.

In some embodiments, provided herein are methods of treating tumor orcancer in a subject in need thereof, comprising administering to thesubject a therapeutically effective amount of the immune effector cells(e.g., BCMA CARTs) disclosed herein. In some embodiments, providedherein are uses of the immune effector cells disclosed herein (e.g.,BCMA CARTs) in treatment of tumor or cancer. In some embodiments,provided herein are uses of the immune effector cells (e.g., BCMA CARTs)provided herein for the preparation of a medicament for the treatment oftumor or cancer. In some embodiments, a population of cells comprisingthe immune effector cell disclosed herein is used in the treatment. Thepopulation of cells can be homogenous. The population of cells can beheterogenous.

In some embodiments, provided herein are methods of treating tumor orcancer in a subject in need thereof, comprising administering to thesubject a therapeutically effective amount of the pharmaceuticalcomposition disclosed herein. In some embodiments, provided herein areuses of the pharmaceutical composition disclosed herein in treatment oftumor or cancer. In some embodiments, provided herein are uses of thepharmaceutical composition provided herein for the preparation of amedicament for the treatment of tumor or cancer.

Actual dosage levels of the active ingredients (i.e., the anti-BCMAantibodies or antigen-binding fragments or the immune effector cellsprovided herein) in the pharmaceutical compositions described herein canbe varied so as to obtain an amount of the active ingredient which iseffective to achieve the desired therapeutic response for a particularpatient, composition, and mode of administration, without being toxic tothe patient. The selected dosage level will depend upon a variety ofpharmacokinetic factors including the activity of the particularcompositions described herein, the route of administration, the time ofadministration, the rate of excretion, the duration of the treatment,other drugs, compounds and/or materials used in combination with theparticular compositions employed, the age, sex, weight, condition,general health and prior medical history of the patient being treated,and like factors well known in the medical arts.

The anti-BCMA antibodies or antigen-binding fragments can beadministered as a sustained release formulation, in which case lessfrequent administration is required. Dosage and frequency vary dependingon the half-life of the anti-BCMA antibodies or antigen-bindingfragments in the patient. In therapeutic applications, a relatively highdosage at relatively short intervals is sometimes required untilprogression of the disease is reduced or terminated, and until thepatient shows partial or complete amelioration of symptoms of disease.

In some embodiments, immune effector cells provided herein thatrecombinantly express the BCMA CARs or TCRs disclosed herein can be usedin the therapeutic methods disclosed herein. When a cell therapy isadopted, the cells provided herein can be administered as a dose basedon cells per kilogram (cells/kg) of body weight of the subject to whichthe cells are administered. The cell doses can be in the range of about10⁴ to about 10¹⁰ cells/kg of body weight, for example, about 10⁵ toabout 10⁹, about 10⁵ to about 10⁸, about 10⁵ to about 10⁷, or about 10⁵to 10⁶ cells/kg of body weight, depending on the mode and location ofadministration. In general, in the case of systemic administration, ahigher dose is used than in regional administration, where the immuneeffector cells are administered in the region of a tumor. The precisedetermination of what would be considered an effective dose can be basedon factors individual to each subject, including their size, age, sex,weight, and condition of the particular subject, as described above.Dosages can be readily determined by those skilled in the art based onthe disclosure herein and knowledge in the art.

The anti-BCMA antibodies or antigen-binding fragments, immune effectorcells, and pharmaceutical compositions provided herein can beadministered to a subject by any methods known in the art, including,but not limited to, pleural administration, intravenous administration,subcutaneous administration, intranodal administration, intratumoraladministration, intramuscular administration, intradermaladministration, intrathecal administration, intrapleural administration,intraperitoneal administration, intracranial administration, spinal orother parenteral routes of administration, for example by injection orinfusion, or direct administration to the thymus. The phrase “parenteraladministration” as used herein means modes of administration other thanenteral and topical administration, usually by injection, and includes,without limitation, intravenous, intramuscular, intraarterial,intrathecal, intracapsular, intraorbital, intracardiac, intradermal,intraperitoneal, transtracheal, subcutaneous, subcuticular,intraarticular, subcapsular, subarachnoid, intraspinal, epidural andintrasternal injection and infusion. In some embodiments, subcutaneousadministration is adopted. In some embodiments, intravenousadministration is adopted. In some embodiments, oral administration isadopted. In one embodiment, the cells provided herein can be deliveredregionally to a tumor using well known methods, including but notlimited to, hepatic or aortic pump; limb, lung or liver perfusion; inthe portal vein; through a venous shunt; in a cavity or in a vein thatis nearby a tumor, and the like. In another embodiment, the cellsprovided herein can be administered systemically. In a preferredembodiment, the cells are administered regionally at the site of atumor. The cells can also be administered intratumorally, for example,by direct injection of the cells at the site of a tumor and/or into thetumor vasculature. For example, in the case of malignant pleuraldisease, mesothelioma or lung cancer, administration is preferably byintrapleural administration (see Adusumilli et al., ScienceTranslational Medicine 6(261):261ra151 (2014)). One skilled in the artcan select a suitable mode of administration based on the type of cancerand/or location of a tumor to be treated. The cells can be introduced byinjection or catheter. In one embodiment, the cells are pleurallyadministered to the subject in need, for example, using an intrapleuralcatheter. Optionally, expansion and/or differentiation agents can beadministered to the subject prior to, during or after administration ofcells to increase production of the cells provided herein in vivo.

Proliferation of the cells provided herein is generally done ex vivo,prior to administration to a subject, and can be desirable in vivo afteradministration to a subject (see Kaiser et al., Cancer Gene Therapy22:72-78 (2015)). Cell proliferation should be accompanied by cellsurvival to permit cell expansion and persistence, such as with T cells.

Diseases that can be treated using the anti-BCMA antibodies orantigen-binding fragments, the immune effector cells, or thepharmaceutical compositions provided herein include any disease ordisorder associated with BCMA, and any disease or disorder in which BCMAis specifically expressed and/or in which BCMA has been targeted fortreatment (collectively “BCMA-associated diseases or disorders”).Cancers associated with BCMA expression include hematologic malignanciessuch as multiple myeloma, Waldenstrom macroglobulinemia, as well as bothHodgkin's and non-Hodgkin's lymphomas. See Coquery et al., Crit RevImmunol., 2012, 32(4):287-305 for a review of BCMA.

In some embodiments, the BCMA-associated disease or disorder is a Bcell-related disorder. In some embodiments, the BCMA-associated diseaseor disorder is glioblastoma, lymphomatoid granulomatosis,post-transplant lymphoproliferative disorder, an immunoregulatorydisorder, heavy-chain disease, primary or immunocyte-associatedamyloidosis, or monoclonal gammopathy of undetermined significance.

In certain diseases and conditions, BCMA is expressed on malignant cellsand cancers. In some embodiments, the cancer is a solid tumor. In someembodiments, the cancer is a hematopoietic cancer. In some embodiments,the cancer is a B cell malignancy. In some embodiments, the cancer is alymphoma, a leukemia, or a plasma cell malignancy. Lymphomascontemplated herein include, but are not limited to, Burkitt lymphoma(e.g., endemic Burkitt's lymphoma or sporadic Burkitt's lymphoma),non-Hodgkin's lymphoma (NHL), Hodgkin's lymphoma, Waldenstrommacroglobulinemia, follicular lymphoma, small non-cleaved cell lymphoma,mucosa-associated lymphatic tissue lymphoma (MALT), marginal zonelymphoma, splenic lymphoma, nodal monocytoid B cell lymphoma,immunoblastic lymphoma, large cell lymphoma, diffuse mixed celllymphoma, pulmonary B cell angiocentric lymphoma, small lymphocyticlymphoma, primary mediastinal B cell lymphoma, lymphoplasmacyticlymphoma (LPL), or mantle cell lymphoma (MCL). Leukemias contemplatedhere, include, but are not limited to, chronic lymphocytic leukemia(CLL), plasma cell leukemia or acute lymphocytic leukemia (ALL).

Also contemplated herein are plasma cell malignancies including, but notlimited to, multiple myeloma (MM) and plasmacytoma.

In some embodiments, the anti-BCMA antibodies or antigen-bindingfragments, the immune effector cells, or the pharmaceutical compositionsprovided herein can be used to treat MM. In some embodiments, the MM tobe treated is non-secretory MM. In some embodiments, the MM issmoldering MM. In some embodiments the disease or condition is relapsedand/or refractory multiple myeloma (R/R MM).

Among the diseases, disorders or conditions associated with BCMA (e.g.,a BCMA-expressing cancer) that can be treated include, but are notlimited to, neuroblastoma, renal cell carcinoma, colon cancer,colorectal cancer, breast cancer, epithelial squamous cell cancer,melanoma, myeloma (e.g., multiple myeloma), stomach cancer, braincancer, lung cancer, pancreatic cancer, cervical cancer, ovarian cancer,liver cancer, bladder cancer, prostate cancer, testicular cancer,thyroid cancer, uterine cancer, adrenal cancer and head and neck cancer.

In some embodiments, the methods include identifying a subject who has,is suspected to have, or is at risk for developing a BCMA-associateddisease or disorder. Hence, provided are methods for identifyingsubjects with diseases or disorders associated with elevated BCMAexpression and selecting them for treatment with the anti-BCMAantibodies or antigen-binding fragments, the immune effector cells, orthe pharmaceutical compositions provided herein.

In some embodiments, a subject can be screened for the presence of adisease or disorder associated with elevated BCMA expression, such as aBCMA-expressing cancer. In some embodiments, the methods includescreening for or detecting the presence of a BCMA-associated disease,e.g., a tumor. Thus, in some embodiments, a sample can be obtained froma patient suspected of having a disease or disorder associated withelevated BCMA expression and assayed for the expression level of BCMA.In some embodiments, a subject who tests positive for a BCMA-associateddisease or disorder can be selected for treatment by the presentmethods, and can be administered a therapeutically effective amount ofthe anti-BCMA antibodies or antigen-binding fragments, the immuneeffector cells, or the pharmaceutical compositions provided herein.

In cancer treatment, eliminating cancer or tumor cells in a subject canoccur, but any clinical improvement constitutes a benefit. An anti-tumoreffect can be manifested by a decrease in tumor volume, a decrease inthe number of tumor cells, a decrease in the number of metastases, anincrease in life expectancy, or amelioration of various physiologicalsymptoms associated with the cancerous condition. An anti-tumor effectcan also be manifested by the ability of the cells or pharmaceuticalcompositions provided herein in prevention of the occurrence of tumor inthe first place. In some embodiments, an “anti-tumor effect” can bemanifested by the reduction in cancer-induced immunosuppression.Clinical improvement comprises decreased risk or rate of progression orreduction in pathological consequences of the cancer or tumor. It isalso understood that a method of treating cancer can include any effectthat ameliorates a sign or symptom associated with cancer. Such signs orsymptoms include, but are not limited to, reducing tumor burden,including inhibiting growth of a tumor, slowing the growth rate of atumor, reducing the size of a tumor, reducing the number of tumors,eliminating a tumor, all of which can be measured using routine tumorimaging techniques well known in the art. Other signs or symptomsassociated with cancer include, but are not limited to, fatigue, pain,weight loss, and other signs or symptoms associated with variouscancers.

In some embodiments, the methods or uses provided herein can reducetumor burden. Thus, administration of the anti-BCMA antibodies orantigen-binding fragments, cells or pharmaceutical compositionsdisclosed herein can reduce the number of tumor cells, reduce tumorsize, and/or eradicate the tumor in the subject. Methods for monitoringpatient response to administration of a pharmaceutical compositiondisclosed herein are known in the art and can be employed in accordancewith methods disclosed herein.

In the methods disclosed herein, a therapeutically effective amount ofthe anti-BCMA antibodies or antigen-binding fragments, cells orpharmaceutical compositions disclosed herein is administered to asubject in need of cancer treatment. The subject can be a mammal. Insome embodiments, the subject is a human. In some embodiments, theseindividuals have no clinically measurable tumor. However, they aresuspected of being at risk for progression of the disease, either nearthe original tumor site, or by metastases. This group can be furthersubdivided into high-risk and low-risk individuals. The subdivision ismade on the basis of features observed before or after the initialtreatment. These features are known in the clinical arts and aresuitably defined for different types of cancers. Features typical ofhigh-risk subgroups are those in which the tumor has invaded neighboringtissues, or who show involvement of lymph nodes.

In some embodiments, the subject has persistent or relapsed disease,e.g., following treatment with another BCMA-specific antibody and/orBCMA CART and/or other therapy, including chemotherapy, radiation,and/or hematopoietic stem cell transplantation (HSCT), e.g., allogeneicHSCT or autologous HSCT. In some embodiments, the administrationeffectively treats the subject despite the subject having becomeresistant to another BCMA-targeted therapy. In some embodiments, thesubject has not relapsed but is determined to be at risk for relapse,such as at a high risk of relapse, and thus the compound or compositionis administered prophylactically, e.g., to reduce the likelihood of orprevent relapse.

In some embodiments, the subject is one that is eligible for atransplant, such as is eligible for a hematopoietic stem celltransplantation (HSCT), e.g., allogeneic HSCT or autologous HSCT. Insome embodiments, the subject has not previously received a transplant,despite being eligible, prior to administration of the BCMA-bindingmolecules, including the anti-BCMA antibodies or antigen-bindingfragments, the immune effector cells, or the pharmaceutical compositionsprovided herein. In some embodiments, the subject is one that is noteligible for a transplant, such as is not eligible for a hematopoieticstem cell transplantation (HSCT), e.g., allogenic HSCT or autologousHSCT.

In some embodiments, the methods provided herein include adoptive celltherapy, whereby genetically engineered immune effector cells expressingthe provided recombinant receptors comprising a BCMA-binding molecule(e.g., BCMA CARs provided herein) are administered to subjects. Suchadministration can promote activation of the cells (e.g., T cellactivation) in a BCMA-targeted manner, such that the cells of thedisease or disorder are targeted for destruction. Thus, the providedmethods and uses include methods and uses for adoptive cell therapy. Insome embodiments, the methods include administration of the cells or acomposition containing the cells to a subject such as one having, or atrisk for, or suspected of having the disease, condition or disorder. Insome embodiments, the cells, populations, and compositions areadministered to a subject having the particular disease or condition tobe treated, e.g., via adoptive cell therapy, such as adoptive T celltherapy. In some embodiments, the cells or compositions are administeredto the subject, such as a subject having or at risk for the disease orcondition. In some aspects, the methods thereby treat, e.g., ameliorateone or more symptom of the disease or condition, such as by lesseningtumor burden in a BCMA-expressing cancer.

Methods for administration of cells for adoptive cell therapy are knownand can be used in connection with the provided methods andcompositions. For example, adoptive T cell therapy methods aredescribed, e.g., in US Patent Application Publication No. 2003/0170238to Gruenberg et al.; U.S. Pat. No. 4,690,915 to Rosenberg; Rosenberg(2011) Nat Rev Clin Oncol. 8(10):577-85). See, e.g., Themeli et al.(2013) Nat Biotechnol. 31(10): 928-933; Tsukahara et al. (2013) BiochemBiophys Res Commun 438(1): 84-9; Davila et al. (2013) PLoS ONE 8(4):e61338.

In some embodiments, the cell therapy, e.g., adoptive cell therapy,e.g., adoptive T cell therapy, is carried out by autologous transfer, inwhich the cells are isolated and/or otherwise prepared from the subjectwho is to receive the cell therapy, or from a sample derived from such asubject. Thus, in some embodiments, the cells are derived from asubject, e.g., patient, in need of a treatment and the cells, followingisolation and processing are administered to the same subject.

In some embodiments, the cell therapy, e.g., adoptive cell therapy,e.g., adoptive T cell therapy, is carried out by allogeneic transfer, inwhich the cells are isolated and/or otherwise prepared from a subjectother than a subject who is to receive or who ultimately receives thecell therapy, e.g., a first subject. In such embodiments, the cells thenare administered to a different subject, e.g., a second subject, of thesame species. In some embodiments, the first and second subjects aregenetically identical. In some embodiments, the first and secondsubjects are genetically similar. In some embodiments, the secondsubject expresses the same HLA class or supertype as the first subject.

Anti-BCMA antibodies or antigen-binding fragments, cells, orpharmaceutical compositions provided herein can be administered withmedical devices known in the art. For example, in some embodiments, aneedleless hypodermic injection device can be used, such as the devicesdisclosed in U.S. Pat. Nos. 5,399,163; 5,383,851; 5,312,335; 5,064,413;4,941,880; 4,790,824; or 4,596,556. Examples of well-known implants andmodules for use described herein include: U.S. Pat. No. 4,487,603, whichdiscloses an implantable micro-infusion pump for dispensing medicationat a controlled rate; U.S. Pat. No. 4,486,194, which discloses atherapeutic device for administering medicaments through the skin; U.S.Pat. No. 4,447,233, which discloses a medication infusion pump fordelivering medication at a precise infusion rate; U.S. Pat. No.4,447,224, which discloses a variable flow implantable infusionapparatus for continuous drug delivery; U.S. Pat. No. 4,439,196, whichdiscloses an osmotic drug delivery system having multi-chambercompartments; and U.S. Pat. No. 4,475,196, which discloses an osmoticdrug delivery system. These patents are incorporated herein byreference. Many other such implants, delivery systems, and modules areknown to those skilled in the art.

Combination therapy using agents with different mechanisms of action canresult in additive or synergetic effects. Combination therapy can allowfor a lower dose of each agent than is used in monotherapy, therebyreducing toxic side effects and/or increasing the therapeutic index ofthe agent disclosed herein. Combination therapy can decrease thelikelihood that resistant cancer cells will develop. In someembodiments, the additional therapy results in an increase in thetherapeutic index of the cells or pharmaceutical compositions describedherein. In some embodiments, the additional therapy results in adecrease in the toxicity and/or side effects of cells or pharmaceuticalcompositions described herein. In some embodiments, the anti-BCMAantibodies or antigen-binding fragments, cells, or pharmaceuticalcompositions described herein can be administered in combination with anadditional therapy. In some embodiments, the additional therapy can besurgical resection, radiotherapy, or chemotherapy.

The additional therapy can be administered prior to, concurrently with,or subsequent to administration of the anti-BCMA antibodies orantigen-binding fragments, cells, or pharmaceutical compositionsdescribed herein. Combined administration can include co-administration,either in a single pharmaceutical formulation or using separateformulations, or consecutive administration in either order butgenerally within a time period such that all active agents can exerttheir biological activities simultaneously. A person skilled in the artcan readily determine appropriate regimens for administering apharmaceutical composition described herein and an additional therapy incombination, including the timing and dosing of an additional agent tobe used in a combination therapy, based on the needs of the subjectbeing treated.

5.8 Methods of Production 5.8.1 Polynucleotides, Polypeptides, andAntibodies

Polynucleotides provided herein can be prepared, manipulated, and/orexpressed using any of the well-established techniques known andavailable in the art. Many vectors can be used. Examples of vectors areplasmid, autonomously replicating sequences, and transposable elements.Exemplary transposon systems such as Sleeping Beauty and PiggyBac can beused, which can be stably integrated into the genome (e.g., Ivics etal., Cell, 91 (4): 501-510 (1997); Cadiñanos et al., (2007) NucleicAcids Research. 35 (12): e87). Additional exemplary vectors include,without limitation, plasmids, phagemids, cosmids, artificial chromosomessuch as yeast artificial chromosome (YAC), bacterial artificialchromosome (BAC), or P1-derived artificial chromosome (PAC),bacteriophages such as lambda phage or M13 phage, and animal viruses.Examples of categories of animal viruses useful as vectors include,without limitation, retrovirus (including lentivirus), adenovirus,adeno-associated virus, herpesvirus (e.g., herpes simplex virus),poxvirus, baculovirus, papillomavirus, and papovavirus (e.g., SV40).Examples of expression vectors are pClneo vectors (Promega) forexpression in mammalian cells; pLenti4N5-DEST™, pLenti6N5-DEST™, andpLenti6.2/V5-GW/lacZ (Invitrogen) for lentivirus-mediated gene transferand expression in mammalian cells.

In some embodiments, the vector is an episomal vector or a vector thatis maintained extrachromosomally. As used herein, the term “episomal”refers to a vector that is able to replicate without integration intohost's chromosomal DNA and without gradual loss from a dividing hostcell also meaning that said vector replicates extrachromosomally orepisomally. The vector is engineered to harbor the sequence coding forthe origin of DNA replication or “ori” from a lymphotrophic herpes virusor a gamma herpesvirus, an adenovirus, SV40, a bovine papilloma virus,or a yeast, specifically a replication origin of a lymphotrophic herpesvirus or a gamma herpesvirus corresponding to oriP of EBV. In someembodiments, the lymphotrophic herpes virus may be Epstein Barr virus(EBV), Kaposi's sarcoma herpes virus (KSHV), Herpes virus saimiri (HS),or Marek's disease virus (MDV). Epstein Barr virus (EBV) and Kaposi'ssarcoma herpes virus (KSHV) are also examples of a gamma herpesvirus.Typically, the host cell comprises the viral replication transactivatorprotein that activates the replication.

“Expression control sequences,” “control elements,” or “regulatorysequences” present in an expression vector are those non-translatedregions of the vector—origin of replication, selection cassettes,promoters, enhancers, translation initiation signals (Shine Dalgarnosequence or Kozak sequence) introns, a polyadenylation sequence, 5′ and3′ untranslated regions—which interact with host cellular proteins tocarry out transcription and translation. Such elements can vary in theirstrength and specificity. Depending on the vector system and hostutilized, any number of suitable transcription and translation elements,including ubiquitous promoters and inducible promoters can be used.

Illustrative ubiquitous expression control sequences that can be used inpresent disclosure include, but are not limited to, a cytomegalovirus(CMV) immediate early promoter, a viral simian virus 40 (SV40) promoter(e.g., early or late), a Moloney murine leukemia virus (MoMLV) LTRpromoter, a Rous sarcoma virus (RSV) LTR, a herpes simplex virus (HSV)(thymidine kinase) promoter, H5, P7.5, and P11 promoters from vacciniavirus, an elongation factor 1-alpha (EF1a) promoter, early growthresponse 1 (EGR1), ferritin H (FerH), ferritin L (FerL), Glyceraldehyde3-phosphate dehydrogenase (GAPDH), eukaryotic translation initiationfactor 4A1 (EIF4A1), heat shock 70 kDa protein 5 (HSPA5), heat shockprotein 90 kDa beta, member 1 (HSP90B1), heat shock protein 70 kDa(HSP70), β-kinesin (β-KIN), the human ROSA 26 locus (Irions et al.,Nature Biotechnology 25, 1477-1482 (2007)), a Ubiquitin C promoter(UBC), a phosphoglycerate kinase-1 (PGK) promoter, a cytomegalovirusenhancer/chicken β-actin (CAG) promoter, and a β-actin promoter.

Illustrative examples of inducible promoters/systems include, but arenot limited to, steroid-inducible promoters such as promoters for genesencoding glucocorticoid or estrogen receptors (inducible by treatmentwith the corresponding hormone), metallothionine promoter (inducible bytreatment with various heavy metals), MX-1 promoter (inducible byinterferon), the “GeneSwitch” mifepristone-regulatable system (Sirin etal., 2003, Gene, 323:67), the cumate inducible gene switch (WO2002/088346), tetracycline-dependent regulatory systems, etc. Theanti-BCMA antibodies or antigen-binding fragments described herein canbe produced by any method known in the art, including chemical synthesisand recombinant expression techniques. The practice of the inventionemploys, unless otherwise indicated, conventional techniques inmolecular biology, microbiology, genetic analysis, recombinant DNA,organic chemistry, biochemistry, PCR, oligonucleotide synthesis andmodification, nucleic acid hybridization, and related fields within theskill of the art. These techniques are described in the references citedherein and are fully explained in the literature. See, e.g., Maniatis etal. (1982) MOLECULAR CLONING: A LABORATORY MANUAL, Cold Spring HarborLaboratory Press; Sambrook et al. (1989), MOLECULAR CLONING: ALABORATORY MANUAL, Second Edition, Cold Spring Harbor Laboratory Press;Sambrook et al. (2001) MOLECULAR CLONING: A LABORATORY MANUAL, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Ausubel etal., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons (1987 andannual updates); CURRENT PROTOCOLS IN IMMUNOLOGY, John Wiley & Sons(1987 and annual updates) Gait (ed.) (1984) OLIGONUCLEOTIDE SYNTHESIS: APRACTICAL APPROACH, IRL Press; Eckstein (ed.) (1991) OLIGONUCLEOTIDESAND ANALOGUES: A PRACTICAL APPROACH, IRL Press; Birren et al. (eds.)(1999) GENOME ANALYSIS: A LABORATORY MANUAL, Cold Spring HarborLaboratory Press; Borrebaeck (ed.) (1995) ANTIBODY ENGINEERING, SecondEdition, Oxford University Press; Lo (ed.) (2006) ANTIBODY ENGINEERING:METHODS AND PROTOCOLS (METHODS IN MOLECULAR BIOLOGY); Vol. 248, HumanaPress, Inc; each of which is incorporated herein by reference in itsentirety.

The polypeptides described herein (e.g., the anti-BCMA antibodies orantigen-binding fragments, or CARs/TCRs) can be produced and isolatedusing methods known in the art. Peptides can be synthesized, in whole orin part, using chemical methods (see, e.g., Caruthers (1980). NucleicAcids Res. Symp. Ser. 215; Horn (1980); and Banga, A. K., THERAPEUTICPEPTIDES AND PROTEINS, FORMULATION, PROCESSING AND DELIVERY SYSTEMS(1995) Technomic Publishing Co., Lancaster, Pa.). Peptide synthesis canbe performed using various solid phase techniques (see, e.g., Roberge,Science 269:202 (1995); Merrifield, Methods. Enzymol. 289:3 (1997)) andautomated synthesis may be achieved, e.g., using the ABI 431A PeptideSynthesizer (Perkin Elmer) in accordance with the manufacturer'sinstructions. Peptides can also be synthesized using combinatorialmethodologies. Synthetic residues and polypeptides can be synthesizedusing a variety of procedures and methodologies known in the art (see,e.g., ORGANIC SYNTHESES COLLECTIVE VOLUMES, Gilman, et al. (Eds) JohnWiley & Sons, Inc., NY). Modified peptides can be produced by chemicalmodification methods (see, for example, Belousov, Nucleic Acids Res.25:3440 (1997); Frenkel, Free Radic. Biol. Med. 19:373 (1995); andBlommers, Biochemistry 33:7886 (1994)). Peptide sequence variations,derivatives, substitutions and modifications can also be made usingmethods such as oligonucleotide-mediated (site-directed) mutagenesis,alanine scanning, and PCR based mutagenesis. Site-directed mutagenesis(Carter et al., Nucl. Acids Res., 13:4331 (1986); Zoller et al., Nucl.Acids Res. 10:6487 (1987)), cassette mutagenesis (Wells et al, Gene34:315 (1985)), restriction selection mutagenesis (Wells et al., Philos.Trans. R. Soc. London SerA 317:415 (1986)) and other techniques can beperformed on cloned DNA to produce invention peptide sequences,variants, fusions and chimeras, and variations, derivatives,substitutions and modifications thereof.

The polypeptides described herein can be prepared using a wide varietyof techniques known in the art including the use of hybridoma andrecombinant technologies, or a combination thereof. In some embodiments,a recombinant expression vector is used to express a polynucleotideencoding a polypeptide described herein. For example, a recombinantexpression vector can be a replicable DNA construct that includessynthetic or cDNA-derived DNA fragments encoding a polypeptideoperatively linked to suitable transcriptional and/or translationalregulatory elements derived from mammalian, microbial, viral or insectgenes. In some embodiments, coding sequences of polypeptides disclosedherein can be ligated into such expression vectors for their expressionin mammalian cells. In some embodiments, a viral vector is used. DNAregions are “operatively linked” when they are functionally related toeach other. For example, a promoter is operatively linked to a codingsequence if it controls the transcription of the sequence; or a ribosomebinding site is operatively linked to a coding sequence if it ispositioned so as to permit translation. In some embodiments, structuralelements intended for use in yeast expression systems include a leadersequence enabling extracellular secretion of translated protein by ahost cell. In some embodiments, in situations where recombinant proteinis expressed without a leader or transport sequence, a polypeptide caninclude an N-terminal methionine residue.

A wide variety of expression host/vector combinations can be employed.Suitable host cells for expression include prokaryotes, yeast cells,insect cells, or higher eukaryotic cells under the control ofappropriate promoters. Appropriate cloning and expression vectors foruse with bacterial, fungal, yeast, and mammalian cellular hosts, as wellas methods of protein production, including antibody production arewell-known in the art. Useful expression vectors for bacterial hostsinclude known bacterial plasmids, such as plasmids from E. coli,including pCR1, pBR322, pMB9 and their derivatives, and wider host rangeplasmids, such as M13 and other filamentous single-stranded DNA phages.

Useful expression vectors for eukaryotic hosts include, for example,vectors comprising expression control sequences from SV40, bovinepapilloma virus, adenovirus, and cytomegalovirus. Examples of suitablemammalian host cell lines include, but are not limited to, COS-7 (monkeykidney-derived), L-929 (murine fibroblast-derived), C127 (murine mammarytumor-derived), 3T3 (murine fibroblast-derived), CHO (Chinese hamsterovary-derived), HeLa (human cervical cancer-derived), BHK (hamsterkidney fibroblast-derived), HEK-293 (human embryonic kidney-derived)cell lines and variants thereof. Mammalian expression vectors cancomprise non-transcribed elements such as an origin of replication, asuitable promoter and enhancer linked to the gene to be expressed, andother 5′ or 3′ flanking non-transcribed sequences, and 5′ or 3′non-translated sequences, such as necessary ribosome binding sites, apolyadenylation site, splice donor and acceptor sites, andtranscriptional termination sequences. Expression of recombinantproteins in insect cell culture systems (e.g., baculovirus) also offersa robust method for producing correctly folded and biologicallyfunctional proteins. Baculovirus systems for production of heterologousproteins in insect cells are well-known to those of skill in the art.

Provided herein are anti-BCMA antibodies and antigen-binding fragmentsthereof that include but are not limited to monoclonal antibodies,polyclonal antibodies, synthetic antibodies, human antibodies, humanizedantibodies, and antigen-binding fragments thereof.

Methods of antibody production are well-known in the art. See forexample, in Harlow et al., Antibodies: A Laboratory Manual, (Cold SpringHarbor Laboratory Press, 2nd ed. 1988); Hammerling et al., in:MONOCLONAL ANTIBODIES AND T-CELL HYBRIDOMAS 563 681 (Elsevier, N.Y.,1981), each of which is incorporated herein by reference in itsentirety. For in vivo use of antibodies in humans, it may be preferableto use human antibodies. Completely human antibodies are particularlydesirable for therapeutic treatment of human subjects. Human antibodiescan be made by a variety of methods known in the art including phagedisplay methods using antibody libraries derived from humanimmunoglobulin sequences, including improvements to these techniques.See, also, U.S. Pat. Nos. 4,444,887 and 4,716,111; and PCT publicationsWO 98/46645, WO 98/50433, WO 98/24893, WO98/16654, WO 96/34096, WO96/33735, and WO 91/10741; each of which is incorporated herein byreference in its entirety. A human antibody can also be an antibodywherein the heavy and light chains are encoded by a nucleotide sequencederived from one or more sources of human DNA.

Human antibodies can also be produced using transgenic mice which areincapable of expressing functional endogenous immunoglobulins, but whichcan express human immunoglobulin genes. For example, the human heavy andlight chain immunoglobulin gene complexes can be introduced randomly orby homologous recombination into mouse embryonic stem cells.Alternatively, the human variable region, constant region, and diversityregion can be introduced into mouse embryonic stem cells in addition tothe human heavy and light chain genes. The mouse heavy and light chainimmunoglobulin genes may be rendered non-functional separately orsimultaneously with the introduction of human immunoglobulin loci byhomologous recombination. For example, it has been described that thehomozygous deletion of the antibody heavy chain joining region (JH) genein chimeric and germ-line mutant mice results in complete inhibition ofendogenous antibody production. The modified embryonic stem cells areexpanded and microinjected into blastocysts to produce chimeric mice.The chimeric mice are then bred to produce homozygous offspring whichexpress human antibodies. The transgenic mice are immunized in thenormal fashion with a selected antigen, e.g., all or a portion of apolypeptide of the invention. For example, anti-BCMA antibodies directedagainst the human BCMA antigen can be obtained from the immunized,transgenic mice using conventional hybridoma technology. The humanimmunoglobulin transgenes harbored by the transgenic mice rearrangeduring B cell differentiation, and subsequently undergo class switchingand somatic mutation. Thus, using such a technique, it is possible toproduce therapeutically useful IgG, IgA, IgM and IgE antibodies,including, but not limited to, IgG1 (gamma 1) and IgG3. For an overviewof this technology for producing human antibodies, see, Lonberg andHuszar (Int. Rev. Immunol., 13:65-93 (1995)). For a detailed discussionof this technology for producing human antibodies and human monoclonalantibodies and protocols for producing such antibodies, see, e.g., PCTPublication Nos. WO 98/24893, WO 96/34096, and WO 96/33735; and U.S.Pat. Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016;5,545,806; 5,814,318; and 5,939,598, each of which is incorporated byreference herein in their entirety. In addition, companies such asAbgenix, Inc. (Freemont, Calif.) and Genpharm (San Jose, Calif.) can beengaged to provide human antibodies directed against a selected antigenusing technology similar to that described above. For a specificdiscussion of transfer of a human germ-line immunoglobulin gene array ingerm-line mutant mice that will result in the production of humanantibodies upon antigen challenge see, e.g., Jakobovits et al, Proc.Natl. Acad. Sci. USA, 90:2551 (1993); Jakobovits et al., Nature,362:255-258 (1993); Bruggermann et al., Year in Immunol., 7:33 (1993);and Duchosal et al., Nature, 355:258 (1992).

Human antibodies can also be derived from phage-display libraries(Hoogenboom et al., J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol.Biol., 222:581-597 (1991); Vaughan et al., Nature Biotech., 14:309(1996)). Phage display technology (McCafferty et al, Nature, 348:552-553(1990)) can be used to produce human antibodies and antibody fragmentsin vitro, from immunoglobulin variable (V) domain gene repertoires fromunimmunized donors. According to this technique, antibody V domain genesare cloned in-frame into either a major or minor coat protein gene of afilamentous bacteriophage, such as M13 or fd, and displayed asfunctional antibody fragments on the surface of the phage particle.Because the filamentous particle contains a single-stranded DNA copy ofthe phage genome, selections based on the functional properties of theantibody also result in selection of the gene encoding the antibodyexhibiting those properties. Thus, the phage mimics some of theproperties of the B cell. Phage display can be performed in a variety offormats; for their review see, e.g., Johnson and Chiswell, CurrentOpinion in Structural Biology 3:564-571 (1993). Several sources ofV-gene segments can be used for phage display. Clackson et al., Nature,352:624-628 (1991) isolated a diverse array of anti-oxazolone antibodiesfrom a small random combinatorial library of V genes derived from thespleens of unimmunized mice. A repertoire of V genes from unimmunizedhuman donors can be constructed and antibodies to a diverse array ofantigens (including self-antigens) can be isolated essentially followingthe techniques described by Marks et al., J. Mol. Biol., 222:581-597(1991), or Griffith et al., EMBO 1, 12:725-734 (1993). See, also, U.S.Pat. Nos. 5,565,332 and 5,573,905, each of which is incorporated hereinby reference in its entirety.

Human antibodies can also be generated by in vitro activated B cells(see, U.S. Pat. Nos. 5,567,610 and 5,229,275, each of which isincorporated herein by reference in its entirety). Human antibodies canalso be generated in vitro using hybridoma techniques such as, but notlimited to, that described by Roder et al. (Methods Enzymol.,121:140-167 (1986)).

Alternatively, in some embodiments, a non-human antibody is humanized,where specific sequences or regions of the antibody are modified toincrease similarity to an antibody naturally produced in a human. Insome embodiment, the antigen binding domain portion is humanized.

A humanized antibody can be produced using a variety of techniques knownin the art, including but not limited to, CDR-grafting (see, e.g.,European Patent No. EP 239,400; International Publication No. WO91/09967; and U.S. Pat. Nos. 5,225,539, 5,530,101, and 5,585,089, eachof which is incorporated herein in its entirety by reference), veneeringor resurfacing (see, e.g., European Patent Nos. EP 592,106 and EP519,596; Padlan, 1991, Molecular Immunology, 28(4/5):489-498; Studnickaet al., 1994, Protein Engineering, 7(6):805-814; and Roguska et al.,1994, PNAS, 91:969-973, each of which is incorporated herein by itsentirety by reference), chain shuffling (see, e.g., U.S. Pat. No.5,565,332, which is incorporated herein in its entirety by reference),and techniques disclosed in, e.g., U.S. Patent Application PublicationNo. US2005/0042664, U.S. Patent Application Publication No.US2005/0048617, U.S. Pat. Nos. 6,407,213, 5,766,886, InternationalPublication No. WO 9317105, Tan et al., J. Immunol., 169:1119-25 (2002),Caldas et al., Protein Eng., 13(5):353-60 (2000), Morea et al., Methods,20(3):267-79 (2000), Baca et al., J. Biol. Chem., 272(16):10678-84(1997), Roguska et al., Protein Eng., 9(10):895-904 (1996), Couto etal., Cancer Res., 55 (23 Supp):5973s-5977s (1995), Couto et al., CancerRes., 55(8):1717-22 (1995), Sandhu J S, Gene, 150(2):409-10 (1994), andPedersen et al., J. Mol. Biol., 235(3):959-73 (1994), each of which isincorporated herein in its entirety by reference. Often, frameworkresidues in the framework regions can be substituted with thecorresponding residue from the CDR donor antibody to alter, preferablyimprove, antigen binding. These framework substitutions are identifiedby methods well-known in the art, e.g., by modeling of the interactionsof the CDR and framework residues to identify framework residuesimportant for antigen binding and sequence comparison to identifyunusual framework residues at particular positions. (See, e.g., Queen etal., U.S. Pat. No. 5,585,089; and Riechmann et al., 1988, Nature,332:323, which are incorporated herein by reference in theirentireties.)

A humanized antibody has one or more amino acid residues introduced intoit from a source which is nonhuman. These nonhuman amino acid residuesare often referred to as “import” residues, which are typically takenfrom an “import” variable domain. Thus, humanized antibodies compriseone or more CDRs from nonhuman immunoglobulin molecules and frameworkregions from human. Humanization of antibodies is well-known in the artand can essentially be performed following the method of Winter andco-workers (Jones et al., Nature, 321:522-525 (1986); Riechmann et al.,Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536(1988)), by substituting rodent CDRs or CDR sequences for thecorresponding sequences of a human antibody, i.e., CDR-grafting (EP239,400; PCT Publication No. WO 91/09967; and U.S. Pat. Nos. 4,816,567;6,331,415; 5,225,539; 5,530,101; 5,585,089; 6,548,640, the contents ofwhich are incorporated herein by reference herein in their entirety). Insuch humanized chimeric antibodies, substantially less than an intacthuman variable domain has been substituted by the corresponding sequencefrom a nonhuman species. In practice, humanized antibodies are typicallyhuman antibodies in which some CDR residues and possibly some FRresidues are substituted by residues from analogous sites in rodentantibodies. Humanization of antibodies can also be achieved by veneeringor resurfacing (EP 592,106; EP 519,596; Padlan, 1991, MolecularImmunology, 28(4/5):489-498; Studnicka et al., Protein Engineering,7(6):805-814 (1994); and Roguska et al., PNAS, 91:969-973 (1994)) orchain shuffling (U.S. Pat. No. 5,565,332), the contents of which areincorporated herein by reference herein in their entirety.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies is to reduce antigenicity. Accordingto the so-called “best-fit” method, the sequence of the variable domainof a rodent antibody is screened against the entire library of knownhuman variable-domain sequences. The human sequence which is closest tothat of the rodent is then accepted as the human framework (FR) for thehumanized antibody (Sims et al., J. Immunol., 151:2296 (1993); Chothiaet al., J. Mol. Biol., 196:901 (1987), the contents of which areincorporated herein by reference herein in their entirety). Anothermethod uses a particular framework derived from the consensus sequenceof all human antibodies of a particular subgroup of light or heavychains. The same framework may be used for several different humanizedantibodies (Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992);Presta et al., J. Immunol., 151:2623 (1993), the contents of which areincorporated herein by reference herein in their entirety).

Antibodies can be humanized with retention of high affinity for thetarget antigen and other favorable biological properties. For example,humanized antibodies can be prepared by a process of analysis of theparental sequences and various conceptual humanized products usingthree-dimensional models of the parental and humanized sequences.Three-dimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, i.e., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind the target antigen. In this way, FR residues canbe selected and combined from the recipient and import sequences so thatthe desired antibody characteristic, such as increased affinity for thetarget antigen, is achieved. In general, the CDR residues are directlyand most substantially involved in influencing antigen binding.

A humanized antibody retains a similar antigenic specificity as theoriginal antibody, for example, the ability to bind human BCMA antigen.However, using certain methods of humanization, the affinity and/orspecificity of binding of the antibody for a particular antigen can beincreased using methods of “directed evolution,” as described by Wu etal., J. Mol. Biol., 294:151 (1999), the contents of which areincorporated herein by reference herein in their entirety.

5.8.2 Genetically Engineered Immune Effector Cells

In some embodiments, provided herein is a genetically engineered immuneeffector cell that comprises a polynucleotide encoding a BCMA CAR or TCRdisclosed herein. In some embodiments, provided herein is a geneticallyengineered immune effector cell that recombinantly expresses a BCMA CARor TCR disclosed herein. In some embodiments, provided herein is agenetically engineered immune effector cell that comprises a vectorcomprising a polynucleotide encoding a BCMA CAR or TCR disclosed herein.In some embodiments, the immune effector cells are T cells.

5.8.2.1 Methods of Genetic Engineering

With respect to generating cells recombinantly expressing a BCMA CAR orTCR disclosed herein, one or more polynucleotides encoding the BCMA CARor TCR is introduced into the target cell using a suitable expressionvector. The target immune effector cells (e.g., T cells) are transferredwith one or more polynucleotides encoding a BCMA CAR or TCR. Thegenetically engineered cells can also express the anti-BCMA antibodiesor antigen-binding fragments disclosed herein.

In some embodiments, provided herein are methods of geneticallyengineering an immune effector cell by transferring a polynucleotideprovided herein into the cell using a non-viral delivery system. TheBCMA CAR or TCR encoding polynucleotide can be mRNA, which allowstransient expression and the self-elimination of the immune effectorcells expressing such BCMA CAR or TCR. Physical methods for introducinga polynucleotide into a host cell include calcium phosphateprecipitation, lipofection, particle bombardment, microinjection,electroporation, and the like. In some embodiments, RNA electroporationcan be used (Van Driessche et al. Folia histochemica et cytobiologica43:4 213-216 (2005)). The methods can further include preparing the mRNAby in vitro transcribing the polynucleotides described herein. In someembodiments, provided herein are methods of genetically engineering animmune effector cell by transferring a polynucleotide encoding anti-BCMAantibodies or antigen-binding fragments disclosed herein into the cellusing electroporation. In some embodiments, provided herein are methodsof genetically engineering an immune effector cell by transferring apolynucleotide encoding BCMA CARs or TCRs disclosed herein into the cellusing electroporation.

In some embodiments, DNA transfection and transposon can be used. Insome embodiments, the Sleeping Beauty system or PiggyBac system is used(e.g., Ivics et al., Cell, 91 (4): 501-510 (1997); Cadiñanos et al.(2007) Nucleic Acids Research. 35 (12): e87). Chemical means forintroducing a polynucleotide into a host cell include colloidaldispersion systems, such as macromolecule complexes, nanocapsules,microspheres, beads, and lipid-based systems including oil-in-wateremulsions, micelles, mixed micelles, and liposomes. An exemplarycolloidal system for use as a delivery vehicle in vitro and in vivo is aliposome (e.g., an artificial membrane vesicle).

For example, a polynucleotide encoding a BCMA CAR or TCR disclosedherein can be cloned into a suitable vector, and introduced into thetarget cell using well known molecular biology techniques (see Ausubelet al., Current Protocols in Molecular Biology, John Wiley and Sons,Baltimore, Md. (1999)). Any vector suitable for expression in a cell,particularly a human cell, can be used. The vectors contain suitableexpression elements such as promoters that provide for expression of theencoded nucleic acids in the target cell.

The use of retroviral vectors for expression in T cells or other immuneeffector cells, including engineered T cells, has been described (seeScholler et al., Sci. Transl. Med. 4:132-153 (2012; Parente-Pereira etal, J. Biol. Methods 1(2):e7 (1-9)(2014); Lamers et al., Blood117(1):72-82 (2011); Reviere et al., Proc. Natl. Acad. Sci. USA92:6733-6737 (1995)). In some embodiments, the vector is a gammaretroviral vector. In one embodiment, the vector is an SGF retroviralvector such as an SGF γ-retroviral vector, which is Moloney murineleukemia-based retroviral vector. SGF vectors have been describedpreviously (see, for example, Wang et al., Gene Therapy 15:1454-1459(2008)). In the case of a retroviral vector, cells can optionally beactivated to increase transduction efficiency (see Parente-Pereira etal., J. Biol. Methods 1(2) e7 (doi 10.14440/jbm.2014.30) (2014);Movassagh et al., Hum. Gene Ther. 11:1189-1200 (2000); Rettig et al.,Mol. Ther. 8:29-41 (2003); Agarwal et al., J. Virol. 72:3720-3728(1998); Pollok et al., Hum. Gene Ther. 10:2221-2236 (1998); Quinn etal., Hum. Gene Ther. 9:1457-1467 (1998); see also commercially availablemethods such as Dynabeads™ human T cell activator products, ThermoFisher Scientific, Waltham, Mass.). It is understood that any suitableviral vector or non-viral delivery system can be used. Combinations of aretroviral vector and an appropriate packaging line are also suitable,where the capsid proteins will be functional for infecting human cells.Various amphotropic virus-producing cell lines are known, including, butnot limited to, PA12 (Miller et al., Mot Cell. Biol. 5:431-437 (1985));PA317 (Miller et al., Mol. Cell. Biol. 6:2895-2902(1986)); and CRIP(Danos et al., Proc. Natl. Acad. Sci. USA 85:6460-6464 (1988)).Non-amphotropic particles are suitable too, for example, particlespseudotyped with VSVG, RD114 or GALV envelope and any other known in theart (Relander et al., Mol. Therap. 11:452-459 (2005)). Possible methodsof transduction also include direct co-culture of the cells withproducer cells (for example, Bregni et al., Blood 80:1418-1422 (1992)),or culturing with viral supernatant alone or concentrated vector stockswith or without appropriate growth factors and polycations (see, forexample, Xu et al., Exp. Hemat. 22:223-230 (1994); Hughes, et al. J.Clin. Invest. 89:1817-1824 (1992)).

Other viral vectors that can be used include, for example, adenoviral,lentiviral, and adeno-associated viral vectors, vaccinia virus, a bovinepapilloma virus derived vector, or a herpes virus, such as Epstein-BarrVirus (see, for example, Miller, Hum. Gene Ther. 1(1):5-14 (1990);Friedman, Science 244:1275-1281 (1989); Eglitis et al., BioTechniques6:608-614 (1988); Tolstoshev et al., Current Opin. Biotechnol. 1:55-61(1990); Sharp, Lancet 337:1277-1278 (1991); Cornetta et al., Prog.Nucleic Acid Res. Mol. Biol. 36:311-322 (1989); Anderson, Science226:401-409 (1984); Moen, Blood Cells 17:407-416 (1991); Miller et al.,Biotechnology 7:980-990 (1989); Le Gal La Salle et al., Science259:988-990 (1993); and Johnson, Chest 107:77S-83S (1995)). Retroviralvectors are particularly well developed and have been used in clinicalsettings (Rosenberg et al., N. Engl. J. Med. 323:370 (1990); Anderson etal., U.S. Pat. No. 5,399,346). Generally, the chosen vector exhibitshigh efficiency of infection and stable expression (see, for example,Cayouette et al., Human Gene Therapy 8:423-430 (1997); Kido et al.,Current Eye Research 15:833-844 (1996); Bloomer et al., J. Virol.71:6641-6649 (1997); Naldini et al., Science 272:263-267 (1996); andMiyoshi et al, Proc. Natl. Acad. Sci. U.S.A. 94:10319-10323 (1997)).

The vectors used herein employ suitable promoters for expression in aparticular host cell. The promoter can be an inducible promoter or aconstitutive promoter. In some embodiments, the promoter of anexpression vector provides expression in a stem cell, such as ahematopoietic stem cell. In some embodiments, the promoter of anexpression vector provides expression in an immune effector cell, suchas a T cell. Non-viral vectors can be used as well, so long as thevector contains suitable expression elements for expression in thetarget cell. Some vectors, such as retroviral vectors, can integrateinto the host genome.

In some embodiments, provided herein are methods of geneticallyengineering an immune effector cell by transferring a polynucleotideprovided herein into the cell using gene-editing. If desired, targetedintegration can be implemented using technologies such as a nuclease,transcription activator-like effector nucleases (TALENs), Zinc-fingernucleases (ZFNs), clustered regularly interspaced short palindromicrepeats (CRISPRs), homologous recombination, non-homologous end joining,microhomology-mediated end joining, homology-mediated end joining andthe like (Gersbach et al., Nucl. Acids Res. 39:7868-7878 (2011);Vasileva, et al. Cell Death Dis. 6:e1831. (Jul. 23, 2015); Sontheimer,Hum. Gene Ther. 26(7):413-424 (2015); Yao et al. Cell Research volume27, 801-814(2017)). In some embodiments, methods provided herein use aZFN system. A zinc-finger nuclease consists of a DNA recognition domainand a non-specific endonuclease. The DNA recognition domain consists ofa series of Cys2-His2 zinc-finger proteins linked in series, and eachzinc-finger unit includes about 30 amino acids for specifically bindingto DNA. The non-specific endonuclease is a FokI endonuclease which formsa dimer to cleave the DNA. In some embodiments, methods provided hereinuse a TALEN system. TALEN is a transcription activator-like effectornuclease. The TALE protein is a core component of a DNA binding domain,and generally consists of a plurality of basic repeat units linked inseries. The designed and combined series of units can specificallyrecognize a DNA sequence and cleave a specific DNA sequence by couplingthe FokI endonuclease.

In some embodiments, methods provided herein use a CRISPR-Cas system.The CRISPR-Cas system can be a CRISPR-Cas9 system. CRISPR/Cas system isa nuclease system consisting of clustered regularly interspaced shortpalindromic repeats (CRISPR) and CRISPR binding proteins (i.e., Casproteins), which can cleave nearly all genomic sequences adjacent toprotospacer-adjacent motifs (PAM) in eukaryocytes (Cong et al. Science2013. 339: 819-823). The “CRISPR/Cas system” is used to refercollectively to transcripts involving CRISPR-related (“Cas”) genes, aswell as other elements involving the expression thereof or directing theactivity thereof, including sequences encoding a Cas gene, tracr(trans-activated CRISPR) sequences (for example, tracrRNA or activepartial tracrRNA), tracr pairing sequences (in the background of anendogenous CRISPR system, cover “direct repeats” and processed partialdirect repeats), guide sequences, or other sequences from the CRISPRlocus and transcripts. In general, the CRISPR system is characterized asan element that facilitates the formation of a CRISPR complex at a siteof a target sequence (also called a protospacer in the endogenous CRISPRsystem). Unrestricted examples of the Cas protein include Cas1, Cas1B,Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 andCsx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2,Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2,Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2,Csf3, Csf4 homologues, or modified forms thereof. In some embodiments,the Cas protein is a Cas9 protein (Gasiunas, Barrangou et al. 2012;Jinek, Chylinski et al. 2012; Deltcheva, Chylinski et al. 2011;Makarova, Grishin et al. (2006)). Amino acid sequences of the Cas9protein are known in the art. Exemplary sequences can be found, forexample, in the SwissProt database under the accession number Q99ZW2, inthe UniProt database under the number A1IQ68, Q03LF7, or J7RUA5.

The vectors and constructs can optionally be designed to include areporter. For example, the vector can be designed to express a reporterprotein, which can be useful to identify cells comprising the vector orpolynucleotides provided on the vector, such as polynucleotides thathave integrated into the host chromosome. In one embodiment, thereporter can be expressed as a bicistronic or multicistronic expressionconstruct with the anti-BCMA antibody or antigen-binding fragment or theBCMA CAR or TCR. Exemplary reporter proteins include, but are notlimited to, fluorescent proteins, such as mCherry, green fluorescentprotein (GFP), blue fluorescent protein, for example, EBFP, EBFP2,Azurite, and mKalama1, cyan fluorescent protein, for example, ECFP,Cerulean, and CyPet, and yellow fluorescent protein, for example, YFP,Citrine, Venus, and YPet.

Assays can be used to determine the transduction efficiency usingroutine molecular biology techniques. If a marker has been included inthe construct, such as a fluorescent protein, gene transfer efficiencycan be monitored by FACS analysis to quantify the fraction of transduced(for example, GFP⁺) immune effector cells, such as T cells, and/or byquantitative PCR. Using a well-established cocultivation system (Gade etal., Cancer Res. 65:9080-9088 (2005); Gong et al., Neoplasia 1:123-127(1999); Latouche et al., Nat. Biotechnol. 18:405-409 (2000)) it can bedetermined whether fibroblast AAPCs expressing cancer antigen (vs.controls) direct cytokine release from transduced immune effector cells,such as T cells, expressing a CAR (cell supernatant LUMINEX (AustinTex.) assay for IL-2, IL-4, IL-10, IFN-γ, TNF-α, and GM-CSF), T cellproliferation (by carboxyfluorescein succinimidyl ester (CFSE)labeling), and T cell survival (by Annexin V staining). The influence ofCD80 and/or 4-1BBL on T cell survival, proliferation, and efficacy canbe evaluated. T cells can be exposed to repeated stimulation by cancerantigen positive target cells, and it can be determined whether T cellproliferation and cytokine response remain similar or diminished withrepeated stimulation. The cancer antigen CAR constructs can be comparedside by side under equivalent assay conditions. Cytotoxicity assays withmultiple E:T ratios can be conducted using chromium-release assays.

Combinations and permutations of various methods described herein orotherwise known in the art are expressly contemplated to prepare thegenetically engineered cells disclosed herein.

5.8.2.2 Manipulation of Immune Effector Cells

Immune effector cells provided herein can be obtained from a subject.Sources for the immune effector cells provided herein include, but arenot limited to, peripheral blood, umbilical cord blood, bone marrow, orother sources of hematopoietic cells. Immune effector cells (e.g., Tcells) can be obtained from a number of sources, including peripheralblood mononuclear cells, bone marrow, lymph node tissue, cord blood,thymus tissue, tissue from a site of infection, ascites, pleuraleffusion, spleen tissue, and tumors. In certain embodiments, cell linesavailable in the art can be used. Immune effector cells provided hereincan be isolated by methods well known in the art, including commerciallyavailable isolation methods (see, for example, Rowland-Jones et al.,LYMPHOCYTES: A PRACTICAL APPROACH, Oxford University Press, New York(1999)). Various methods for isolating immune effector cells have beendescribed previously, and can be used, including but not limited to,using peripheral donor lymphocytes (Sadelain et al., Nat. Rev. Cancer3:35-45 (2003); Morgan et al., Science 314: 126-129 (2006), and usingselectively in v/Yro-expanded antigen-specific peripheral bloodleukocytes employing artificial antigen-presenting cells (AAPCs) ordendritic cells (Dupont et al., Cancer Res. 65:5417-5427 (2005);Papanicolaou et al., Blood 102:2498-2505 (2003)).

In certain embodiments, immune effector cells (e.g., T cells) disclosedherein can be obtained from a unit of blood collected from a subjectusing any techniques known to the skilled artisan, such as Ficoll™separation. In some embodiments, cells from the circulating blood of anindividual are obtained by apheresis. The apheresis product typicallycontains lymphocytes, including T cells, monocytes, granulocytes, Bcells, other nucleated white blood cells, red blood cells, andplatelets. In some embodiments, the cells collected by apheresis can bewashed to remove the plasma fraction and to place the cells in anappropriate buffer or media for subsequent processing steps. In someembodiments, the cells are washed with phosphate buffered saline (PBS).In an alternative embodiment, the wash solution lacks calcium and maylack magnesium or may lack many if not all divalent cations. Initialactivation steps in the absence of calcium lead to magnified activation.As those of ordinary skill in the art would readily appreciate a washingstep can be accomplished by methods known to those in the art, such asby using a semi-automated “flow-through” centrifuge (for example, theCobe 2991 cell processor, the Baxter CytoMate, or the Haemonetics CellSaver 5) according to the manufacturer's instructions. After washing,the cells can be resuspended in a variety of biocompatible buffers, suchas, for example, Ca²⁺-free, Mg²⁺-free PBS, PlasmaLyte A, or other salinesolution with or without buffer. Alternatively, the undesirablecomponents of the apheresis sample can be removed, and the cellsdirectly resuspended in culture media.

In another embodiment, T cells are isolated from peripheral bloodlymphocytes by lysing the red blood cells and depleting the monocytes,for example, by centrifugation through a PERCOLL™ gradient or bycounterflow centrifugal elutriation. A specific subpopulation of Tcells, such as CD3⁺, CD28⁺, CD4⁺, CD8⁺, CD45RA⁺, and CD45RO⁺ T cells,can be further isolated by positive or negative selection techniques.For example, in one embodiment, T cells are isolated by incubation withanti-CD3/anti-CD28 (i.e., 3×28)-conjugated beads, such as DYNABEADS®M-450 CD3/CD28 T, for a time period sufficient for positive selection ofthe desired T cells. In one embodiment, the time period is about 30minutes. In a further embodiment, the time period ranges from 30 minutesto 36 hours or longer and all integer values there between. In a furtherembodiment, the time period is at least 1, 2, 3, 4, 5, or 6 hours. Inyet another preferred embodiment, the time period is 10 to 24 hours. Inone preferred embodiment, the incubation time period is 24 hours. Forisolation of T cells from patients with leukemia, use of longerincubation times, such as 24 hours, can increase cell yield. Longerincubation times may be used to isolate T cells in any situation wherethere are few T cells as compared to other cell types, such in isolatingtumor infiltrating lymphocytes (TIL) from tumor tissue or fromimmune-compromised individuals. Further, use of longer incubation timescan increase the efficiency of capture of CD8+ T cells. Thus, by simplyshortening or lengthening the time T cells are allowed to bind to theCD3/CD28 beads and/or by increasing or decreasing the ratio of beads toT cells (as described further herein), subpopulations of T cells can bepreferentially selected for or against at culture initiation or at othertime points during the process. Additionally, by increasing ordecreasing the ratio of anti-CD3 and/or anti-CD28 antibodies on thebeads or other surface, subpopulations of T cells can be preferentiallyselected for or against at culture initiation or at other desired timepoints. The skilled artisan would recognize that multiple rounds ofselection can also be used in the context of this invention.

Various techniques can be employed to separate the cells to enrich fordesired immune effector cells. For instance, negative selection methodscan be used to remove cells that are not the desired immune effectorcells. Additionally, positive selection methods can be used to isolateor enrich for desired immune effector cells or precursor cells thereof,or a combination of positive and negative selection methods can beemployed. Monoclonal antibodies (MAbs) are particularly useful foridentifying markers associated with particular cell lineages and/orstages of differentiation for both positive and negative selections. Ifa particular type of cell is to be isolated, for example, a particulartype of T cell, various cell surface markers or combinations of markers,including but not limited to, CD3, CD4, CD8, CD34 (for hematopoieticstem and progenitor cells) and the like, can be used to separate thecells, as is well known in the art (see Kearse, T CELL PROTOCOLS:DEVELOPMENT AND ACTIVATION, Humana Press, Totowa N.J. (2000); De Libero,T CELL PROTOCOLS, Vol. 514 of Methods in Molecular Biology, HumanaPress, Totowa N.J. (2009)). In some embodiments, enrichment of a T cellpopulation by negative selection can be accomplished with a combinationof antibodies directed to surface markers unique to the negativelyselected cells. One method is cell sorting and/or selection via negativemagnetic immunoadherence or flow cytometry that uses a cocktail ofmonoclonal antibodies directed to cell surface markers present on thecells negatively selected. For example, to enrich for CD4⁺ cells bynegative selection, a monoclonal antibody cocktail typically includesantibodies to CD14, CD20, CD11b, CD16, HLA-DR, and CD8. In certainembodiments, it may be desirable to enrich for or positively select forregulatory T cells which typically express CD4⁺, CD25⁺, CD62L^(hi),GITR⁺, and FoxP3⁺. Alternatively, in certain embodiments, T regulatorycells are depleted by anti-C25 conjugated beads or other similar methodof selection.

Procedures for separation of immune effector cells include, but are notlimited to, density gradient centrifugation, coupling to particles thatmodify cell density, magnetic separation with antibody-coated magneticbeads, affinity chromatography; cytotoxic agents joined to or used inconjunction with a monoclonal antibody (mAb), including, but not limitedto, complement and cytotoxins, and panning with an antibody attached toa solid matrix, for example, a plate or chip, elutriation, flowcytometry, or any other convenient technique (see, for example,Recktenwald et al., CELL SEPARATION METHODS AND APPLICATIONS, MarcelDekker, Inc., New York (1998)). It is understood that the immuneeffector cells used in methods provided herein can be substantially purecells or can be a polyclonal population. In some embodiments, apolyclonal population can be enriched for a desired immune effectorcell. Such an enrichment can take place prior to or after geneticallyengineering the cells to express a BCMA CAR or TCR provided herein, asdesired.

The immune effector cells can be autologous or non-autologous to thesubject to which they are administered in the methods of treatmentdisclosed herein. Autologous cells are isolated from the subject towhich the engineered cells are to be administered. Optionally, the cellscan be obtained by leukapheresis, where leukocytes are selectivelyremoved from withdrawn blood, made recombinant, and then retransfusedinto the donor. Alternatively, allogeneic cells from a non-autologousdonor that is not the subject can be used. In the case of anon-autologous donor, the cells are typed and matched for humanleukocyte antigen (HLA) to determine an appropriate level ofcompatibility, as is well known in the art. The cells can optionally becryopreserved after isolation and/or genetic engineering, and/orexpansion of genetically engineered cells (see Kaiser et al., supra,2015)). Methods for cyropreserving cells are well known in the art (see,for example, Freshney, CULTURE OF ANIMAL CELLS: A MANUAL OF BASICTECHNIQUES, 4th ed., Wiley-Liss, New York (2000); Harrison and Rae,GENERAL TECHNIQUES OF CELL CULTURE, Cambridge University Press (1997)).

In some embodiments, isolated immune effector cells are geneticallyengineered ex vivo for recombinant expression of a polypeptide (e.g.,CAR or TCR). In some embodiments, isolated immune effector cells aregenetically engineered ex vivo for recombinant expression of a BCMA CARor TCR. In some embodiments, immune effector cells provided herein areobtained by in vitro sensitization, wherein the sensitization can occurbefore or after the immune effector cells are genetically engineered torecombinantly express a polypeptide disclosed herein. In an embodimentwhere the sensitized immune effector cells, such T cells, are isolatedfrom in vivo sources, it will be self-evident that genetic engineeringoccurs of the already-sensitized immune effector cells.

Also contemplated in the present disclosure is the collection of bloodsamples or apheresis product from a subject at a time period prior towhen the genetically engineered cells as described herein might beneeded. As such, the source of the cells to be expanded can be collectedat any time point necessary, and desired cells, such as T cells,isolated and frozen for later use in T cell therapy for any number ofdiseases or conditions that would benefit from T cell therapy, such asthose described herein. In one embodiment, a blood sample or anapheresis is taken from a generally healthy subject. In certainembodiments, a blood sample or an apheresis is taken from a generallyhealthy subject who is at risk of developing a disease, but who has notyet developed a disease, and the cells of interest are isolated andfrozen for later use. In certain embodiments, the T cells can beexpanded, frozen, and used at a later time. In certain embodiments,samples are collected from a patient shortly after diagnosis of aparticular disease as described herein but prior to any treatments. In afurther embodiment, the cells are isolated from a blood sample or anapheresis from a subject prior to any number of relevant treatmentmodalities, including but not limited to treatment with agents such asnatalizumab, efalizumab, antiviral agents, chemotherapy, radiation,immunosuppressive agents, such as cyclosporin, azathioprine,methotrexate, mycophenolate, and FK506, antibodies, or otherimmunoablative agents such as CAMPATH, anti-CD3 antibodies, cytoxan,fludarabine, cyclosporin, FK506, rapamycin, mycophenolic acid, steroids,FR901228, and irradiation. These drugs inhibit either the calciumdependent phosphatase calcineurin (cyclosporine and FK506) or inhibitthe p70S6 kinase that is important for growth factor induced signaling(rapamycin) (Liu et al, Cell 66:807-815, 1991; Henderson et al., Immun73:316-321, 1991; Bierer et al, Curr. Opin. Immun. 5:763-773, 1993). Ina further embodiment, the cells are isolated for a patient and frozenfor later use in conjunction with (e.g., before, simultaneously orfollowing) bone marrow or stem cell transplantation, T cell ablativetherapy using either chemotherapy agents such as, fludarabine,external-beam radiation therapy (XRT), cyclophosphamide, or antibodiessuch as OKT3 or CAMPATH. In another embodiment, the cells are isolatedprior to and can be frozen for later use for treatment following B-cellablative therapy such as agents that react with CD20, e.g., Rituxan.

In a further embodiment, T cells are obtained from a patient directlyfollowing treatment. In this regard, it has been observed that followingcertain cancer treatments, in particular treatments with drugs thatdamage the immune system, shortly after treatment during the period whenpatients would normally be recovering from the treatment, the quality ofT cells obtained can be optimal or improved for their ability to expandex vivo. Likewise, following ex vivo manipulation using the methodsdescribed herein, these cells may be in a preferred state for enhancedengraftment and in vivo expansion. Thus, it is contemplated to collectblood cells, including T cells, NK cells, or other immune effector cellsof the hematopoietic lineage, during this recovery phase. Further, incertain embodiments, mobilization (for example, mobilization withGM-CSF) and conditioning regimens can be used to create a condition in asubject wherein repopulation, recirculation, regeneration, and/orexpansion of particular cell types is favored, especially during adefined window of time following therapy. Illustrative cell typesinclude T cells, B cells, dendritic cells, and other cells of the immunesystem.

The immune effector cells disclosed herein can be subjected toconditions that favor maintenance or expansion of cells as well known inthe art. (De Libero, T Cell Protocols, Vol. 514 of Methods in MolecularBiology, Humana Press, Totowa N.J. (2009); Parente-Pereira et al., J.Biol. Methods 1(2) e7 (doi 10.14440/jbm.2014.30) (2014); Movassagh etal., Hum. Gene Ther. 11:1189-1200 (2000); Rettig et al., Mol. Ther.8:29-41 (2003); Agarwal et al., J. Virol. 72:3720-3728 (1998); Pollok etal., Hum. Gene Ther. 10:2221-2236 (1999); Quinn et al., Hum. Gene Ther.9:1457-1467 (1998); see also commercially available methods such asDynabeads™ human T cell activator products, Thermo Fisher Scientific,Waltham, Mass.)). The immune effector cells disclosed herein (e.g., Tcells) can optionally be expanded prior to or after ex vivo geneticengineering. Expansion of the cells is particularly useful to increasethe number of cells for administration to a subject. Such methods forexpansion of cells are well known in the art (see e.g., Kaiser et al.,Cancer Gene Therapy 22:72-78 (2015); Wolfl et al., Nat. Protocols9:950-966 (2014)). Furthermore, the cells can optionally becryopreserved after isolation and/or genetic engineering, and/orexpansion of genetically engineered cells (see Kaiser et al., supra,2015)). Methods for cyropreserving cells are well known in the art (see,for example, Freshney, Culture of Animal Cells: A Manual of BasicTechniques, 4th ed., Wiley-Liss, New York (2000); Harrison and Rae,General Techniques of Cell Culture, Cambridge University Press (1997)).

Generally, the T cells provided herein can be expanded by contact with asurface having attached thereto an agent that stimulates a CD3/TCRcomplex associated signal and a ligand that stimulates a co-stimulatoryreceptor on the surface of the T cells. In particular, T cellpopulations can be stimulated as described herein, such as by contactwith an anti-CD3 antibody, or antigen-binding fragment thereof, or ananti-CD2 antibody immobilized on a surface, or by contact with a proteinkinase C activator (e.g., bryostatin) in conjunction with a calciumionophore. For co-stimulation of an accessory molecule on the surface ofthe T cells, a ligand that binds the accessory molecule is used. Forexample, a population of T cells can be contacted with an anti-CD3antibody and an anti-CD28 antibody, under conditions appropriate forstimulating proliferation of the T cells. To stimulate proliferation ofeither CD4⁺ T cells or CD8⁺ T cells, an anti-CD3 antibody and ananti-CD28 antibody. Examples of an anti-CD28 antibody include 9.3, B-T3,XR-CD28 (Diaclone, Besancon, France) can be used as can other methodscommonly known in the art (Berg et al., Transplant Proc.30(8):3975-3977, 1998; Haanen et al, J. Exp. Med. 190(9):13191328, 1999;Garland et al., J. Immunol Meth. 227(1-2):53-63, 1999).

The invention is generally disclosed herein using affirmative languageto describe the numerous embodiments. The invention also specificallyincludes embodiments in which particular subject matter is excluded, infull or in part, such as substances or materials, method steps andconditions, protocols, procedures, assays or analysis. Thus, even thoughthe invention is generally not expressed herein in terms of what theinvention does not include, aspects that are not expressly included inthe invention are nevertheless disclosed herein.

Particular embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Upon reading the foregoing description, variations of the disclosedembodiments shall become apparent to individuals working in the art, andit is expected that those skilled artisans can employ such variations asappropriate. Accordingly, it is intended that the invention be practicedotherwise than as specifically described herein, and that the inventionincludes all modifications and equivalents of the subject matter recitedin the claims appended hereto as permitted by applicable law. Moreover,any combination of the above-described elements in all possiblevariations thereof is encompassed by the invention unless otherwiseindicated herein or otherwise clearly contradicted by context.

All publications, patent applications, accession numbers, and otherreferences cited in this specification are herein incorporated byreference in its entirety as if each individual publication or patentapplication were specifically and individually indicated to beincorporated by reference. The publications discussed herein areprovided solely for their disclosure prior to the filing date of thepresent application. Nothing herein is to be construed as an admissionthat the present invention is not entitled to antedate such publicationby virtue of prior invention. Further, the dates of publication providedcan be different from the actual publication dates which need to beindependently confirmed.

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, the descriptions in the Experimental are intended toillustrate but not limit the scope of invention described in the claims.

5.9 Experimental

As disclosed in detail below, twelve novel anti-BCMA scFv were generatedand characterized. T cells expressing CARs that comprise these BCMA scFvwere also generated and characterized. The cytotoxicity of these BCMACARTs against BCMA-expressing tumors was confirmed.

5.9.1 Materials and Methods

Primary human lymphocytes. Primary human CD4+ T cells and CD8+ T cellswere isolated from healthy volunteer donors. T cells were stimulatedwith anti-CD3/CD28 Dynabeads (Life Technologies, Grand Island, N.Y.) andcultured in the R10 medium (RPMI-1640 medium supplemented with 10% fetalbovine serum, 1% HEPES, 1% GutaMAX, 1% penicillin and streptomycin, 1%MEM NEAA, and 1% sodium pyruvate) supplemented with 100 IU/mL IL-2.

Cell lines. The following cell lines were cultured in R10 medium andused in related experiments: Nalm6 (human B-cell acute lymphoblasticleukemia cells), Jeko1 (human lymphoma cells), RPMI-8226 (human myelomacells), Raji (human lymphoma cells), THP1 (human leukemia cells), HCC70(human breast cancer cells), 786-O (human renal cell carcinoma cells),SH-SY5Y (human neuroblastoma cells), A549ESO (human lung cancer cells),SKOV3 (human ovarian cancer cells), PC3 (human prostate cancer cells),H226 (human lung carcinoma cells), ASPC1 (human pancreatic tumor cells),Caski (human cervical cancer cells), and K562 (human leukemia cells).

Lentivirus production and transduction. Lentiviral vectors were producedfrom HEK293T cells transfected with transfer plasmids and packagingplasmids pRSV.REV, pMD2.G and pMDLg.pRRE. Lentiviral vectors wereharvested after 24 and 48 hours and concentrated by ultracentrifugation.

T cells (CD4:CD8=1:1) were stimulated by CD3/CD28 dynabeads on day 0.Lentivirus was added to culture medium on day 1. The cells were fed withR10 medium with 100 IU/ml IL-2 every day or every two days.

Production of BCMA31.BBz and LACO mRNA. In vitro transcription of mRNAwas performed using the Ambion mMessage mMACHINE T7 Ultra kit (LifeTechnologies, Carlsbad, Calif.).

Electroporation of BCMA31.BBz and LACO mRNA. mRNA was added to 0.1 ml Tcells (1×108 cells/ml) that were washed twice and resuspended withOPTI-MEM. Electroporation was performed in a 0.2 cm cuvette with aBTX830 (Harvard Apparatus BTX) at 500 V and 0.7 ms.

Tumor killing assay using IncuCyte real-time cell analyzer. Tumor cellsand T cells were washed with R10 medium twice, and then were resuspendedin R10 medium. Both tumor cells and CAR+ T cells were seeded at 10,000cells/well in a 96 well flat-bottom plate. Total integrated intensitywas recorded every 4 hours.

ELISA. 0.1 million of CAR-T cells and 0.1 million of tumor cells wereco-cultured at 37° C. for 24 h, then the supernatant was collected andthe ELISA assay was performed to measure IL-2 and IFNγ secretion levelsfollowing the instruction manual of ELISA kits (R&D Systems).

CD107a assay. CAR-T cells and tumor cells were mixed at a ratio of 1:2.CD107 antibody was added to the medium. One hour after coculture,GolgiStop solution was added. After 3 more hours, the cells were stainedby CD3-BV421 and CD8-APC antibodies and were analyzed by flow cytometry.

5.9.2 Preparation of Anti-BCMA Antibodies

Anti-BCMA antibodies were prepared using fully human antibody phagedisplay library following the steps below:

(1) Expression and purification of phage display library: the log phaseTG1 library culture was infected with freshly thawed M13K07 helper phagewith a multiplicity of infection of 20:1 (phage-to-cell-ratio) andovernight induction by IPTG; the phage library was purified by PEG/NaClprecipitated method and phage titer was determined. The phage was storedat 4° C. and the scFv selection was performed shortly after.

(2) Selection of BCMA-specific scFv-phages: for the first round ofselection, Maxisorp plate was coated with 20 μg/ml BCMA-6His proteindissolved in 1×PBS and incubated overnight at 4° C. (For subsequentrounds of selection, lower protein concentration was used for morestringent selection, including 2 in the 2nd round bio-panning, and 0.5μg/ml in the 3rd round bio-panning.) The plate was then washed threetimes with PBS, blocking buffer (5% milk+1% BSA in 1×PBS) was added toeach well. After 2-hour incubation at room temperature, the blockingbuffer was discarded, phage solution added, and the plate was sealedwith parafilm, and incubated for 2 hours with gently shaking. In thefirst selection round, the plate was then washed 10 times with PBST.(For following rounds, increase stringency of washing was adopted byadding more wash cycles: 20 cycles in the 2nd round, 30 cycles in the3rd round). The antigen-bound scFv-phages were then eluted using 1 mlacid elution buffer (pH 2.2), neutralized, inoculated in 15 ml oflog-phase TG1 culture (OD600=0.5), cultured at 37° C. by 30 min standingand 30 min shaking, plated onto 2×YT-GA agar plate, and culturedovernight at 30° C. for subsequent selection.

(3) mpELISA screening: after three round selection, 480 phage-infectedbacterial colonies were selected for monoclonal phage ELISA (mpELISA)screening. Phage supernatant was generated from individual bacterialclones and tested for the binding to BCMA-Fc protein. The supernatantwas incubated with pre-blocked Maxisorp plate coated with 2 μg/mlBCMA-6His protein. After three washes, 100 μl/well of HRP-conjugatedanti-M13 antibody diluted 1:5000 in blocking buffer (5% milk+1% BSA in1×PBS) was added and incubate for 60 min at RT. After washing plate 5times with PBST, 100 μl/well TMB substrate solution was added andincubated for 10-30 min until blue color had appeared. Reaction wasstopped by adding 50 μl/well of stop solution (2N H2SO4). Absorbance wasread at 450 nm in a microplate reader. Table 4 below provides the readsof three representative 96-well plate of anti-human BCMA-Fc monoclonalphage ELISA. Positive clones are those with gray highlights. A total of36 positive colonies in the phage ELISA test were selected to amplifythe scFv fragment by PCR and sent to sequencing.

TABLE 4 1 2 3 4 5 6 7 8 9 10 11 12 Plate-1 A 0.013 0.011 0.011 0.0212.017 0.009 0.015 0.013 2.062 0.006 0.01 0.016 B 2.31 2.505 0.009 0.010.01 0.007 0.009 0.009 0.006 0.009 0.012 0.009 C 0.055 1.886 0.009 0.0110.01 0.007 0.021 0.008 0.004 0.006 0.016 0.009 D 0.009 0.01 2.031 1.1680.009 0.072 0.008 0.007 0.006 0.005 0.009 0.006 E 0.01 0.011 0.01 1.8250.01 0.006 0.009 0.01 0.007 0.013 0.009 0.057 F 0.009 0.011 0.009 0.010.009 0.007 0.009 0.008 1.027 2.512 0.006 0.011 G 0.009 0.009 0.009 0.010.008 2.453 0.008 0.007 2.325 0.875 0.008 0.01 H 0.029 −0.002 0.014 0.010.089 0.008 0.028 0.009 0.028 0.037 0.014 0.078 Plate-2 A 0.012 0.0090.015 0.009 0.01 0.011 0.014 0.013 0.009 0.009 0.015 0.043 B 1.669 2.1630.013 0.007 0.007 0.004 0.008 0.006 0.006 0.005 0.004 0.014 C 0.0070.009 0.007 0.006 0.006 0.004 0.005 0.006 0 0.001 0 −0.001 D 0.009 0.0070.008 0.005 0.005 0.024 0.008 0.004 0.002 0 1.749 0.006 E 0.145 0.0090.009 1.606 0.01 0.006 0.005 0.007 0.002 0.003 0.005 0.007 F 0.01 0.0130.409 0.007 0.01 0.004 0.005 0.006 0.002 0.001 0.005 0.006 G 0.038 0.010.013 2.276 0.008 2.138 0.008 0.008 0.006 0.006 0.015 0.011 H 0.0170.121 0.011 0.012 0.015 0.011 2.32 0.012 0.009 0.007 0.01 0.026 Plate-3A 0.011 0.008 0.009 0.006 0.007 0.006 0.82 0.01 0.007 0.005 0.007 0.156B 0.008 0.008 0.007 0.007 0.007 0.005 0.01 0.008 1.631 0.006 0.008 0.008C 0.008 0.009 0.008 1.526 0.008 0.004 0.008 0.007 1.764 0.002 0.0070.007 D 2.223 0.007 0.013 0.005 0.982 0.001 0.005 0.005 0.001 0.0010.003 1.992 E 0.006 0.007 0.005 0.005 0.005 0.002 0.006 0.005 0.0030.004 2.691 0.005 F 0.005 0.007 0.006 0.006 0.006 0.003 0.004 0.0050.002 0.003 2.582 0.01 G 0.006 0.007 0.009 0.008 0.003 0 0.004 0.0050.005 0.054 0.004 0.003 H 0.012 0.009 0.007 0.007 0.011 0.002 0.0071.158 2.219 0.004 0.256 0.008

(4) Cloning and sequence analysis: positive clones were selectedaccording to the ELISA results and used as templates for PCR cloning ofthe scFv sequence (Forward primer sequence: tgcagctggcacgacaggtttc (SEQID NO: 25), reverse primer sequence: cgtcagactgtagcacgtt (SEQ ID NO:26)). The PCR products were then sequenced by sanger sequencing method(Forward primer sequence: aacaattgaattcaggagga (SEQ ID NO: 27), reverseprimer sequence: cctcctaagaagcgtagtc (SEQ ID NO: 28)). The CDR regionsof scFv were analyzed through abysis website (http://abysis.org/) andare provided above in Tables 1 and 2.

(5) Screening of functional anti-BCMA scFv(s) in T cell: anti-BCMAscFv(s) were constructed into a bicistronic lentiviral CAR expressionvector, which contained an IRES-truncated EGFR (tEGFR) expressingcassette. Lentivirus was generated by transient transfection in 293Tcells, then purified and concentrated by ultra-centrifuge. T cells weretransduced with CAR lentivirus to generate CAR-T cells, and cultured foranother 10 days. 10 days after lentivirus-transduction, CAR-T cells werecollected and stained with 5 μg/ml CD19-Fc protein (Ctrl Fc protein) orBCMA-Fc recombinant protein at 4° C. for 30 min. After washing, theCAR-T cells were stained with anti-human IgG Fc and anti-EGFR mAb.Sample was analyzed by flow cytometry. As shown, T cells expressing CARscomprising the following anti-BCMA scFv(s) showed binding to BCMA-Fc(FIG. 1B) and were selected for further studies: BCMA21, BCMA22, BCMA23,BCMA24, BCMA27, BCMA28, BCMA30, BCMA31, BCMA32, BCMA33, BCMA34, andBCMA35.

5.9.3 Preparation and Characterization of BCMA CART

We constructed 12 different anti-BCMA CARs using the anti-BCMA scFv(s)described above. Three other CART products were tested in parallel,including NBC10 (Novartis AG and University of Pennsylvania,BMCA10.BBz), FHVH33 (National Institutes of Health, US), and B38M(Nanjing Legend Biotech). All tested CARs had a 41BBz coactivationdomain.

TABLE 5 BCMA CARTs, CAR %, and Expression Levels CART scFv CAR % MFI 1NBC10 22% 1.2E+05 2 FHVH33 84% 3.2E+05 3 BCMA21 31% 1.6E+05 4 BCMA22 12%7.5E+04 5 BCMA23 18% 2.2E+05 6 BCMA24 23% 9.4E+04 7 BCMA27 27% 1.7E+05 8BCMA28 29% 6.5E+04 9 BCMA30 25% 4.6E+04 10 BCMA31 37% 2.5E+05 11 BCMA3219% 5.6E+04 12 BCMA33 27% 2.6E+05 13 BCMA34 16% 9.5E+04 14 BCMA35 18%1.1E+05 15 B38M 51% 5.8E+05 16 NTD

T cells were transduced by lentiviral vectors to express different BCMACARs. Table 5 above shows the CART cells used in the studies disclosedherein, the percentage of the CAR-expressing cells, and their respectiveexpression levels. FIGS. 2A and 2B show the frequencies of CAR+ T cellsand their expression levels (Mean Fluorescence Intensity; “MFI”),respectively. Among the twelve scFv we generated, BCMA31 (#10; SEQ IDNO:15) and BCMA33 (#12) were expressed at higher levels than the rest.FIG. 3 shows comparable frequencies of CAR+CD8 cells among tested CARTs.FIG. 4 shows the phenotypes of CART cells. The frequencies of naïve Tcell population (CD45RO−; CCR7+) in BCMA27 (#7), BCMA31 (#10) and BCMA33(#12) T cells were higher than those in other samples, indicating theseT cells were less differentiated.

5.9.4 Expression of BCMA by Tumor Cells

As shown in FIGS. 5A and 5B, different tumor cell lines were examinedfor the expression of BCMA by FACS staining (FIG. 5A) and RT-PCR (FIG.5B). BCMA expression was detected in Jeko-1 (low level), Raji(intermediate level) and RPMI-8226 cells (high level) by FACS staining.Although BCMA expression was not detected in Nalm6 by FACS, RT-PCRanalysis showed it was expressed, albeit at a very low level.

5.9.5 BCMA CART Showed Cytotoxicity Against Tumor Cells

The CART cells were cocultured with Jeko-1 cells and RPMI-8226 tumorcells. The production of INF-γ and IL-2 were examined. As shown in FIGS.6A (INF-γ) and 6B (IL-2), of the 12 CARTs that we generated, BCMA23(#5), BCMA24 (#6), BCMA27 (#7), BCMA31 (#10; SEQ ID NO:15), and BCMA33(#12) produced more cytokines than the others, including NBC10 and B38MCART cells.

We also examined the cytolytic activities of the CART cells againstJeko-1 (FIGS. 7A-7D) and RPMI-8226 cells (FIGS. 8A-8E), respectively.BCMA23 (#5), BCMA24 (#6), BCMA31 (#10; SEQ ID NO: 15), and BCMA33 (#12)CART cells showed different levels of cytotoxicity against Jeko-1 cells,with BCMA31 (#10) being the highest, which efficiently eliminatedJeko-1. Additionally, BCMA21 (#3), BCMA22 (#4), BCMA23 ( ), BCMA24 (#6),BCMA27 (#7), BCMA31 (#10; SEQ ID NO:15), BCMA33 (#12), BCMA4 (#13), andBCMA35 (#14) showed different levels of cytotoxicity against RPMI-8226cells, of which BCMA21 (#3), BCMA23 (#5), BCMA24 (#6), BCMA27 (#7),BCMA31 (#10), and BCMA33 (#12) efficiently eliminated RPMI-8226 cells.

What is claimed is:
 1. An antibody or antigen-binding fragment thereofthat specifically binds BCMA, comprising: (a) a light chain variableregion (VL) comprising a light chain CDR1 (VL CDR1), a light chain CDR2(VL CDR2), and a light chain CDR3 (VL CDR3) having an amino acidsequence of SEQ ID NOs: 1, 2, and 3 respectively; or a variant thereofhaving up to about 5 amino acid substitutions, additions, and/ordeletions in the VL CDRs; and/or (b) a heavy chain variable region (VH)comprising a heavy chain CDR1 (VH CDR1), a heavy chain CDR2 (VH CDR2),and a heavy chain CDR3 (VH CDR3) having an amino acid sequence of SEQ IDNOs: 4, 5, and 6 respectively; or a variant thereof having up to about 5amino acid substitutions, additions, and/or deletions in the VH CDRs. 2.The antibody or antigen-binding fragment of claim 1, comprising a VLCDR1, a VL CDR2, a VL CDR3, a VH CDR1, a VH CDR2 and a VH CDR3, wherein(a) the VL CDR1, CDR2 and CDR3 have the amino acid sequences of SEQ IDNOs:1, 2, and 3, respectively; and (b) the VH CDR1, CDR2 and CDR3 havethe amino acid sequences of SEQ ID NOs:4, 5, and 6, respectively.
 3. Theantibody or antigen-binding fragment of claim 1, comprising: (a) a VLhaving at least 85% sequence identity to an amino acid sequence of SEQID NO:7; and/or (b) a VH having at least 85% sequence identity to anamino acid sequence of SEQ ID NO:8.
 4. The antibody or antigen-bindingfragment of claim 1, comprising a VL and a VH, wherein the VL and VHhave the amino acid sequences of SEQ ID NO:7 and 8 respectively.
 5. Theantibody or antigen-binding fragment of claim 1 that is a monoclonalantibody or antigen-binding fragment.
 6. The antibody or antigen-bindingfragment of claim 1 that is selected from the group consisting of anIgG1 antibody, an IgG2 antibody, an IgG3 antibody, and an IgG4 antibody.7. The antibody or antigen-binding fragment of claim 1 that is selectedfrom the group consisting of a Fab, a Fab′, a F(ab′)₂, a Fv, a scFv, a(scFv)₂, a single domain antibody (sdAb), and a heavy chain antibody(HCAb).
 8. The antibody or antigen-binding fragment of claim 1 that is achimeric antibody or antigen-binding fragment, a humanized antibody orantigen-binding fragment, or a human antibody or antigen-bindingfragment.
 9. A Chimeric Antigen Receptor (CAR) that specifically bindsBCMA, comprising, from N-terminus to C-terminus: (a) a BCMA-bindingdomain that comprises the antibody or antigen-binding fragment of claim1; (b) a transmembrane domain; and (c) a cytoplasmic domain.
 10. The CARof claim 9, wherein the transmembrane domain is derived from CD8, CD28,CD3ζ, CD4, 4-1BB, OX40, ICOS, CTLA-4, PD-1, LAG-3, 2B4, BTLA, TCR αchain, TCR β chain, or TCR ζ chain, CD3ε, CD45, CD5, CD8, CD9, CD16,CD22, CD33, CD37, CD64, CD80, CD86, CD134, or CD154.
 11. The CAR ofclaim 9, wherein the transmembrane domain comprises CD8 transmembraneregion or CD28 transmembrane region.
 12. The CAR of claim 9, wherein thecytoplasmic domain comprises a signaling domain derived from CD3ζ, FcRγ,FcγRIIa, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, DAP10, DAP12,or any combination thereof.
 13. The CAR of claim 9, wherein thecytoplasmic domain further comprises a co-stimulatory domain derivedfrom CD28, 4-1BB (CD137), OX40, ICOS, DAP10, 2B4, CD27, CD30, CD40, CD2,CD7, LIGHT, GITR, TLR, DR3, CD43, or any combination thereof.
 14. TheCAR of claim 9, wherein the cytoplasmic domain comprises a CD3ζsignaling domain and a 4-1BB co-stimulatory domain, or the cytoplasmicdomain comprises a CD3ζ signaling domain and a CD28 co-stimulatorydomain.
 15. The CAR of claim 9, further comprising a CD8 hinge betweenthe antibody or antigen-binding fragment and the transmembrane domain.16. The CAR of claim 9 comprising an amino acid sequence of SEQ IDNO:15.
 17. A cell comprising the polynucleotide encoding CAR of claim 9.18. The cell of claim 17 that is an immune effector cell.
 19. The cellof claim 17 that is derived from a cell isolated from peripheral bloodor bone marrow.
 20. The cell of claim 17 that is derived from a celldifferentiated in vitro from a stem or progenitor cell selected from thegroup consisting of a T cell progenitor cell, a hematopoietic stem andprogenitor cell, a hematopoietic multipotent progenitor cell, anembryonic stem cell, and an induced pluripotent cell.
 21. The cell ofclaim 17 that is a T cell or a NK cell.
 22. The cell of claim 17 that isa cytotoxic T cell, a helper T cell, a gamma delta T, a CD4+/CD8+ doublepositive T cell, a CD4+ T cell, a CD8+ T cell, a CD4/CD8 double negativeT cell, a CD3+ T cell, a naive T cell, an effector T cell, a helper Tcell, a memory T cell, a regulator T cell, a Th0 cell, a Th1 cell, a Th2cell, a Th3 (Treg) cell, a Th9 cell, a Th17 cell, a Thαβ helper cell, aTfh cell, a stem memory TSCM cell, a central memory TCM cell, aneffector memory TEM cell, or an effector memory TEMRA cell.
 23. A methodof treating cancer in a subject in need thereof, comprisingadministering to the subject a therapeutically effective amount of theantibody or antigen-binding fragment of claim
 1. 24. The method of claim23, further comprising administering an additional therapy to thesubject.
 25. The method of claim 23, wherein the subject is a human. 26.The method of claim 23, wherein the cancer is a BCMA-expressing cancer.27. The method of claim 23, wherein the cancer is a solid tumor or ahematological cancer.
 28. The method of claim 23, wherein the cancer isa B cell malignancy.
 29. The method of claim 23, wherein the cancer is alymphoma, a leukemia, or a plasma cell malignancy.
 30. The method ofclaim 23, wherein the caner is multiple myeloma (MM), Waldenstrommacroglobulinemia, Hodgkin's lymphoma or non-Hodgkin's lymphoma.